Compositions and methods for treating cancer and modulating stress granule formation

ABSTRACT

The invention provides methods for treating or decreasing the likelihood of developing a stress-granule related disorder and/or cancer by administering one or more poly-ADP-ribose polymerase (PARP) inhibitors, one or more PARP activators, one or more poly-ADP-ribose glycosylase (PARG) activators, and/or one or more poly-ADP-ribose glycohydrolase ARH3 activators. The invention also provides corresponding methods of decreasing stress granule formation and/or proliferation in a cell or a population of cells. The invention further provides methods of increasing the number of stress granules and proliferation in a cell or a population of cells by administering one or more PARP activators, one or more PARP inhibitors, one or more PARG inhibitors, and/or one or more ARH3 inhibitors. The invention also provides methods for screening for agents for treating or decreasing the likelihood of developing a stress granule-related disorder or cancer, and methods for determining the propensity for developing a stress granule-related disorder or cancer, as well as compositions and kits containing one or more PARP inhibitors, one or more PARP activators, one or more PARG activators, and one or more ARH3 activators.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 61/269,614, filed Jun. 26, 2009, hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology andmolecular medicine.

BACKGROUND OF THE INVENTION

Poly-adenosine diphosphate (ADP)-ribose (PAR) polymers are the productof post-translational modifications carried out by PAR polymerases(PARPs). PAR is polymerized by PARPs onto acceptor proteins usingnicotinamide adenine dinucleotide (NAD⁺) as substrate (FIG. 1). PARpolymers are localized to distinct cellular structures in differentphases of the cell cycle and localize to the mitotic spindle duringmitosis (FIG. 2). There are at least 18 PARPs in the human genome: thedomain structure for several PARPs is depicted in FIG. 3. However, thespecific biological function and protein substrates of these PARPs arenot fully characterized (Ame et al., Bioessays 26:882-893, 2004). Theidentification of the function and the substrates of each member of thisfamily of proteins has been difficult to date.

PAR polymers are required for normal cell division and PARP knockouts inDrosophila melanogaster are embryonic lethal (Tulin et al., Genes Dev.16:2108-2119, 2002). The concentration, length, and extent of PARbranching are regulated by a balance of activities of the PARPs and PARglycohydrolase (PARG), a highly specific, processive endo- andexo-glycosidase (Hatakeyama et al., J. Biol. Chem. 261:14902-14911,1986). Poly-ADP-ribose polymers have generally been implicated for arole in several different human diseases including cancer, ischemicinjury, inflammatory diseases, cardiovascular diseases, andneurodegenerative disorders.

We have discovered that several PARP proteins are localized to thenucleus and/or are required for cell cycle progression through mitosis.We have also discovered a role for several PARP proteins in theformation, nucleation, and disassembly of stress granules. Stressgranules are distinct cellular structures that form in the cytosol uponexposure of a cell to stress conditions. Stress granules are composed ofboth proteins and RNA molecules. The RNA molecules present in stressgranules are mRNA molecules stalled in translation pre-initiationcomplexes. Stress granules are typically 100 to 200 nM in size and arecommonly associated with the endoplasmic reticulum. Stress granules havebeen implicated in several different disease states includingcardiovascular disorders, inflammatory disorders, neurologicaldisorders, and ischemic-reperfusion injury.

Methods and compositions for the treatment of stress granule-relateddisorders and cancer are presently desired.

SUMMARY OF THE INVENTION

The invention provides methods of treating or decreasing (e.g., by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) thelikelihood of developing a stress granule-related disorder in a subjectrequiring administering to a subject a therapeutically effective amountof one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARPinhibitor(s), one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)poly-ADP ribose glycosylase (PARG) activators, and/or one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP11 activators. The invention alsoprovides methods for decreasing the number of stress granules present ina cell or in a population of cells requiring contacting the cell orpopulation of cells with an effective amount of one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) PARP inhibitor(s), one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) PARG activators, and/or one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP11 activators. The invention alsoprovides compositions and kits containing one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) PARP inhibitor(s), one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) PARG activator(s), and/or one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) PARP11 activators.

In each of the above of the above methods, compositions, and kits, theone or more PARP inhibitor(s) may selectively decrease (e.g., at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100%) theexpression (e.g., mRNA or protein) and/or activity of one or more (e.g.,1, 2, 3, 4, or 5) of PARP5a, PARP12, PARP13 isoform 1 (PARP13.1), PARPisoform 2 (PARP13.2), and PARP15. In different embodiments of the aboveaspects of the invention, the decrease in expression of one or more ofPARP5a, PARP12, PARP13.1, PARP13.2, or PARP15 is a decrease in the levelof one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid(s)containing a nucleic acid sequence having at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) sequence identity toPARP5a (SEQ ID NO: 8 or 9), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO:19), PARP13.2 (SEQ ID NO: 20), or PARP15 (SEQ ID NO: 22 or 23), or adecrease in the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) polypeptides encoded by these nucleic acids. In differentembodiments of the above aspects, the activity of the one or more ofPARP5a, PARP12, PARP13.1, PARP13.2, or PARP15 is poly-ADP-ribosylationof one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) targetprotein(s) (e.g., a protein localized in a stress granule, a polypeptideinvolved in the formation or disassembly of a stress granule, and/or aPARP protein) or formation and/or nucleation of a stress granule.

In each of the above aspects, the one or more PARG activators mayselectively increase (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, or 100%) the expression (e.g., mRNA and/or protein)and/or one or more activities of PARG protein or poly-ADP-riboseglycohydrolase ARH3. In different embodiments of the above aspects ofthe invention, the increase in expression of PARG or ARH3 is an increasein the level of one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleic acid(s)containing a nucleic acid sequence having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARG(SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41), or an increase in the level ofone or more (e.g., 1, 2, 3, 4, 5, or 6) polypeptides encoded by thesenucleic acids. In different embodiments of the above aspects, the one ormore (e.g., 1, 2, 3, 4, or 5) activities of PARG or ARH3 is hydrolysisof poly-ADP-ribose (e.g., poly-ADP-ribose attached to one or more (e.g.,1, 2, 3, 4, or 5) substrate protein(s), e.g., a protein localized in astress granule, a polypeptide involved in the formation or disassemblyof a stress granule, and/or a PARP protein), the prevention of theassembly of a stress granule, or disassembly of a stress granule.

In each of the above of the above methods, compositions, and kits, theone or more PARP11 activator (s) may selectively increase (e.g., atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even100%) the expression (e.g., mRNA and/or protein) and/or one or more(e.g., 1, 2, 3, 4, or 5) activities of PARP11. In different embodimentsof the above aspects of the invention, the increase in expression ofPARP11 is an increase in the level of one or more (e.g., 1, 2, 3, 4, or5) nucleic acid(s) containing a nucleic acid sequence having at least80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%,or even 100%) to PARP11 (SEQ ID NO: 17), or an increase in the level ofone or more (e.g., 1, 2, 3, 4, or 5) polypeptides encoded by thesenucleic acids. In different embodiments of the above aspects, the one ormore (e.g., 1, 2, 3, 4, or 5) activities of PARP11 ispoly-ADP-ribosylation of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) target protein(s) (e.g., a protein localized in a stress granule,a polypeptide involved in the formation or disassembly of a stressgranule, and/or a PARP protein), the prevention of the assembly of astress granule, or the disassembly of a stress granule.

In each of the above aspects, the one or more PARP inhibitors may be anantibody or antibody fragment that selectively binds one or more (e.g.,1, 2, 3, 4, or 5) of PARP5a, PARP12, PARP13.1, PARP13.2, and PARP15; anRNA aptamer (e.g., one or more RNA aptamers containing the sequence ofone of SEQ ID NOS: 40, 49, 99-113, and 122-129); or a small molecule. Ineach of the above aspects, the one or more PARG activators may be one ormore (e.g., 1, 2, 3, 4, or 5) nucleic acid(s) containing a nucleic acidsequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100%) to PARG (SEQ ID NO: 42) or ARH3(SEQ ID NO: 41). In each of the above aspects, the one or more PARP11activators may be one or more (e.g., 1, 2, 3, 4, or 5) nucleic acid(s)containing a nucleic acid sequence having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) toPARP11 (SEQ ID NO: 17).

In each of the above methods of treatment, the one or more PARPinhibitor(s), one or more PARG activator(s), and/or one or more PARP11activators may be administered once a day or one or more times a week(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times a week),and/or administered parenterally (e.g., intravenous, intraarterial,subcutaneous, or intramuscular administration) or orally, and/oradministered with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)additional therapeutic agents (e.g., one or more (e.g., 1, 2, 3, 4, or5) non-steroidal anti-inflammatory drug(s), one or more (e.g., 1, 2, 3,4, or 5) immunosuppressive agent(s), one or more (e.g., 1, 2, 3, 4, or5) calcineurin inhibitors, and/or one or more (e.g., 1, 2, 3, 4, or 5)analgesic(s)). In each of the above methods of treating a cell or cellpopulation, the contacting may result in a reduction in the number ofstress granules in a cell (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or even 100% reduction in the number of stress granulesin a cell or cell population compared to a control cell or cellpopulation, e.g., a cell or cell population from a person having ordiagnosed with a stress granule-related disorder).

The invention further provides methods of treating or decreasing (e.g.,by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) thelikelihood of developing cancer in a subject requiring administering toa subject a therapeutically effective amount of one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) PARP inhibitor(s). The invention alsoprovides methods for decreasing (e.g., at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or even 100%) proliferation of a cell or apopulation of cells requiring contacting the cell or population of cellswith an effective amount of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10) PARP inhibitor(s). The invention also provides compositionsand kits containing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)PARP inhibitor(s).

In each of these methods, compositions, and kits, the one or more PARPinhibitor(s) may selectively decrease (e.g., at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% decrease) theexpression (e.g., mRNA and/or protein) and/or one or more (e.g., 1, 2,3, 4, or 5) activities of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8)of PARP1, PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16. Indifferent embodiments of these aspects of the invention, the decrease inexpression of the one or more of PARP1, PARP2, PARP5A, PARP5B, PARP7,PARP8, PARP14, and PARP16 is a decrease in the level of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid(s) containing anucleic acid sequence having at least 80% sequence identity (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARP1 (SEQ IDNO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B(SEQ ID NO: 10), PARP14 (SEQ ID NO: 21), or PARP16 (SEQ ID NO: 24), or adecrease in the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) polypeptides encoded by these nucleic acids. In differentembodiments of these aspects of the invention, the one or moreactivities of the one or more of PARP1, PARP2, PARP5A, PARP5B, PARP7,PARP8, PARP14, or PARP16 is poly-ADP-ribosylation of one or more (e.g.,1, 2, 3, 4, or 5) target protein(s) (e.g., a protein localized in thenucleus or mitotic spindle during cytokinesis, and/or a PARP protein) oris required for progression through mitosis.

In each of the these aspects, the one or more PARP inhibitors may be anantibody or antibody fragment that selectively binds one or more (e.g.,1, 2, 3, 4, 5, 6, 7, or 8) of PARP1, PARP2, PARP5A, PARP5B, PARP7,PARP8, PARP14, and PARP16; an RNA aptamer (e.g., an RNA aptamercontaining the sequence of one or more of SEQ ID NOS: 43-46, 49, 50,59-74, 114-121, and 130-136); or a small molecule. In each of thesemethods of treatment, the one or more PARP inhibitor(s) may beadministered once a day or one or more times a week (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times a week), and/or administeredparenterally (e.g., intravenous, intraarterial, subcutaneous, orintramuscular administration) or orally, and/or administered with one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional therapeuticagents (e.g., one or more (e.g., 1, 2, 3, 4, or 5) chemotherapeuticagents, one or more (e.g., 1, 2, 3, 4, or 5) non-steroidalanti-inflammatory drug(s), one or more (e.g., 1, 2, 3, 4, or 5)immunosuppressive agent(s), one or more (e.g., 1, 2, 3, 4, or 5)calcineurin inhibitors, and/or one or more (e.g., 1, 2, 3, 4, or 5)analgesic(s)). In each of the these methods of treating a cell or cellpopulation, the contacting may result in a reduction in the rate ofproliferation of a cell or cell population (e.g., by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% reduction in the rate ofproliferation of a cell or cell population compared to a control cell orcell population, e.g., a cancer cell or a cancer cell line).

The invention further provides methods for increasing (e.g., by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%) the number ofstress granules in a cell or cell population by contacting the cell orcell population with an effective amount of one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10) PARP activators, one or more (e.g., 1, 2, 3, 4,or 5) PARG inhibitors, and/or one or more (e.g., 1, 2, 3, 4, or 5)PARP11 inhibitors. In one embodiment of these methods, the one or morePARP activators selectively increase the expression (e.g., mRNA and/orprotein) and/or one or more (e.g., 1, 2, 3, 4, or 5) activities of oneor more (e.g., 1, 2, 3, 4, or 5) of PARP5A, PARP12, PARP13.1, PARP13.2,and PARP15. In different embodiments of these methods, the increase inexpression is an increase (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or even 100% increase) in the level of one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid(s) containing a nucleicacid sequence having at least 80% sequence identity (e.g., at least 85%,90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARP5A (SEQ ID NO: 8 or9), PARP5b (SEQ ID NO: 10), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ IDNO: 20), or PARP15 (SEQ ID NO: 22 or 23), or an increase in the level ofone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptidesencoded by these nucleic acids. In different embodiments of this method,the one or more activities of PARP5A, PARP12, PARP13.1, PARP13.2, andPARP15 is the poly-ADP-ribosylation of one or more (e.g., 1, 2, 3, 4, or5) target protein(s) (e.g., a protein localized in a stress granule, aprotein involved in the formation or disassembly of a stress granule,and/or a PARP protein) or the formation or nucleation of a stressgranule.

In different embodiments of this method, the one or more PARG inhibitorsmay selectively decrease (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or even 100%) the expression (e.g., mRNA and/or protein)and/or one or more (e.g., 1, 2, 3, 4, or 5) activities of PARG or ARH3.In additional embodiments of this method, the decrease in expression ofPARG or ARH3 is a decrease in the level of one or more (e.g., 1, 2, 3,4, 5, or 6) nucleic acid(s) containing a nucleic sequence having atleast 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or even 100%) to PARG (SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41),or a decrease in the levels of one or more (e.g., 1, 2, 3, 4, 5, or 6)polypeptides encoded by these nucleic acids. In additional embodimentsof this method, the one or more activities of PARG or ARH3 is thehydrolysis of poly-ADP-ribose (e.g., poly-ADP-ribose that is covalentlyattached to one or more (e.g., 1, 2, 3, 4, or 5) substrate protein(s),e.g., a protein localized in a stress granule, a protein involved in theformation or disassembly of a stress granule, and/or a PARP protein),the prevention of assembly of a stress granule protein, or disassemblyof a stress granule.

In an additional embodiment of this method, the one or more PARP11inhibitors may selectively decrease (e.g., at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or even 100%) the expression (e.g., mRNA and/orprotein) and/or one or more activities of PARP11. In differentembodiments of this method, the decrease (e.g., at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or even 100%) in expression of PARP11 is adecrease in the level of one or more (e.g., 1, 2, 3, 4, or 5) nucleicacid(s) containing a nucleic acid having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) toPARP11 (SEQ ID NO: 17), or a decrease in the level of one or more (e.g.,1, 2, 3, 4, or 5) polypeptides encoded by these nucleic acids. Indifferent embodiments of this method, the one or more activities ofPARP11 is poly-ADP-ribosylation of one or more (e.g., 1, 2, 3, 4, or 5)target protein(s) (e.g., a protein localized in a stress granule, aprotein involved in the formation or disassembly of a stress granule,and/or a PARP protein), the prevention of the assembly of a stressgranule, or disassembly of a stress granule.

In additional embodiments of this method, the one or more PARPactivators is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)nucleic acid(s) comprising a nucleic acid sequence having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100%) to PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10),PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO: 20), or PARP15 (SEQ IDNO: 22 or 23). In additional embodiments of this method, the one or morePARG inhibitors is an antibody or antibody fragment that selectivelybinds to PARG or ARH3; an RNA aptamer (e.g., a nucleic acid sequencethat contains one or more of SEQ ID NOS: 34-37); or a small molecule. Inadditional embodiments of this method, the one or more PARP11 inhibitorsmay be an antibody or an antibody fragment that selectively binds toPARP11; an RNA aptamer (e.g., a nucleic acid sequence that contains oneor more of SEQ ID NOS: 91-98); or a small molecule.

In additional embodiments of this method, the contacting results in atleast a 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100%) reduction in the number of stress granules present in thecell or the population of cells compared to a control cell or populationof cells (e.g., a cell or population of cells untreated with a PARPactivator, a PARG inhibitor, or a PARP11 inhibitor, e.g., a cell from asubject having or diagnosed with a stress-granule disorder). Inadditional embodiments of this method, the cell or population of cellsis a mammalian cell(s) or a plant cell(s).

The invention also provides methods of increasing (e.g., by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%) theproliferation of a cell or a population of cells requiring contactingthe cell or population of cells with an effective amount of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP activators. In differentembodiments of this method, the one or more PARP activators selectivelyincrease (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100%) the expression (e.g., mRNA and/or protein) and/or one or moreactivities (e.g., 1, 2, 3, 4, or 5) of one or more (e.g., 1, 2, 3, 4, 5,6, 7, or 8) of PARP1, PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, andPARP16. In additional embodiments of this method, the increase inexpression of one or more of PARP1, PARP2, PARP5A, PARP5B, PARP7, PARP8,PARP14, or PARP16 is an increase in the level of one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid(s) comprising a nucleic acidsequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100%) to PARP1 (SEQ ID NO: 1 or 2),PARP2 (SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO:10), PARP14 (SEQ ID NO: 21), or PARP16 (SEQ ID NO: 24), or an increasein the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)polypeptides encoded by these nucleic acids. In additional embodimentsof this method, the one or more activities of said one or more of PARP1,PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, or PARP16 ispoly-ADP-ribosylation of one or more (e.g., 1, 2, 3, 4, or 5) targetprotein(s) (e.g., a protein localized in the nucleus or mitotic spindleduring cytokinesis, and/or a PARP protein) or is required forprogression through mitosis.

In additional embodiments of this method, the one or more PARPactivators is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)nucleic acid(s) containing a nucleic acid sequence having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100%) to PARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP5A(SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP14 (SEQ ID NO: 21), orPARP16 (SEQ ID NO: 24). In different embodiments of this method, thecell or cell population is a plant cell(s) or mammalian cell(s). Indifferent embodiments of this method, the contacting results in at leasta 10% increase (e.g., at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or even 100% increase) in the rate of proliferation of the cell orpopulation of cells compared to a control cell or cell population (e.g.,a cell or cell population not treated with a PARP activator).

The invention further provides methods for identifying a candidate agentfor treating or decreasing (e.g., by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95%) the likelihood of developing a stressgranule-related disorder requiring the steps of: providing one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/orARH3 fusion proteins encoded by a nucleic acid containing a nucleic acidsequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100%) to PARP5A (SEQ ID NO: 8 or 9),PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO:20), PARP15 (SEQ ID NO: 22 or 23), PARG (SEQ ID NO: 42), or ARH3 (SEQ IDNO: 41); contacting the one or more PARP, PARG, and/or ARH3 proteins,and/or PARP, PARG, and/or ARH3 fusion proteins with the agent and alabeled nicotinamide adenine dinucleotide (NAD⁺) substrate; andmeasuring one or more (e.g., 1, 2, 3, 4, or 5) activities of the one ormore PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3fusion proteins, or the specific binding of the agent to said one ormore PARP proteins and/or PARP fusion proteins; wherein an agent thatdecreases (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95%) the one or more activities and/or specifically binds to theone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or16) PARP proteins and/or PARP fusion proteins, and/or increases theactivity of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) PARGand/or ARH3 proteins, and/or PARG and/or ARH3 fusion proteins isidentified as a candidate agent for treating or decreasing thelikelihood of developing a stress granule-related disorder.

The invention further provides methods for identifying a candidate agentfor treating or decreasing (e.g., by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95%) the likelihood of developing cancerrequiring the steps of providing one or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16) PARP protein(s) and/or PARP fusionprotein(s) encoded by a nucleic acid containing a nucleic acid sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or even 100%) to PARP1 (SEQ ID NO: 1 or 2), PARP2(SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10),PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP14 (SEQ ID NO: 21), orPARP16 (SEQ ID NO: 24); contacting the one or more PARP protein(s)and/or PARP fusion protein(s) with the agent and a labeled NAD⁺substrate; and measuring the one or more (e.g., 1, 2, 3, 4, or 5)activities of the one or more PARP protein(s) and/or PARP fusionprotein(s), and/or the specific binding of the agent to the one or morePARP protein(s) and/or PARP fusion protein(s); wherein an agent thatdecreases (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95%) the one or more activities and/or specifically binds to the oneor more PARP protein(s) and/or fusion protein(s) is identified as acandidate for treating or reducing the likelihood of developing cancer.

In different embodiments of the screening methods, the labeled NAD⁺substrate is labeled with a radioisotope (e.g., ³²P) or fluorophore, oris biotinylated. In different embodiments of the screening methods, theone or more PARP, PARG, and/or ARH3 protein(s) and/or PARP, PARG, and/orARH3 fusion protein(s) is purified. In another embodiment of thescreening methods, the one or more PARP, PARG, and/or ARH3 protein(s)and/or PARP, PARG, and/or ARH3 fusion protein(s) is present in a celllysate. In different embodiments of the screening methods, the agent isa small molecule (e.g., a small molecule from a chemical library), apolypeptide or peptide fragment (e.g., a polypeptide or peptide fragmentpresent in a cellular lysate), or a nucleic acid. In additionalembodiments of the above screening methods, the one or more PARP, PARG,and/or ARH3 protein(s) and/or PARP, PARG, and/or ARH3 fusion protein(s)is attached to a substrate (e.g., a magnetic bead) or a solid surface.In additional embodiments of the above screening methods, the method isperformed in a multi-well plate.

The invention further provides methods for determining the propensity ofa subject to develop a stress granule-related disorder by determiningthe expression and/or one or more (e.g., 1, 2, 3, 4, or 5) activities ofone or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of PARP5A, PARP11, PARP12,PARP13.1, PARP13.2, PARP15, PARG, and ARH3 in a subject, wherein anincrease (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%) in the expression (e.g., mRNA and/or protein) and/or one or more(e.g., 1, 2, 3, 4, or 5) activities of one or more (e.g., 1, 2, 3, 4, or5) of PARP5A, PARP12, PARP13.1, PARP13.2, and PARP15, and/or a decrease(e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even100%) in the expression (e.g., mRNA and/or protein) or one or more(e.g., 1, 2, 3, 4, or 5) activities of one or more (e.g., 1, 2, or 3) ofPARP11, PARG, or ARH3 indicates an increased propensity to develop astress granule-related disorder. In an additional embodiment of thismethod, the expression is the level of one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) nucleic acid(s) containing a nucleic acid sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or even 100%) to PARP5A (SEQ ID NO: 8 or 9), PARP5B(SEQ ID NO: 10), PARP11 (SEQ ID NO: 17), PARP13.1 (SEQ ID NO: 19),PARP13.2 (SEQ ID NO: 20), PARP15 (SEQ ID NO: 20 or 23), PARG (SEQ ID NO:42), or ARH3 (SEQ ID NO: 41), or the level of one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) polypeptides encoded by these nucleic acids.

The invention also provides a method for determining the propensity of asubject to develop cancer comprising determining the expression (e.g.,mRNA and/or protein) and/or one or more (e.g., 1, 2, 3, 4, or 5)activities of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of PARP1,PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16 in a subject,wherein an increase in the expression and/or one or more activities ofone or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16 indicates an increasedpropensity to develop cancer. In an additional embodiment of thismethod, the expression is the level of one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) nucleic acid(s) containing a nucleic acid sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or even 100%) to PARP1 (SEQ ID NO: 1 or 2), PARP2(SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10),PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP14 (SEQ ID NO: 21), orPARP16 (SEQ ID NO: 24), or the level of one or more polypeptides (e.g.,1, 2, 3, 4, 5, 6, 7, or 8) encoded by these nucleic acids.

In any of the above screening methods, the expression of the one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid(s) is determinedusing reverse transcriptase polymerase chain reaction (RT-PCR). Inadditional embodiments of the above screening methods, the expression ofthe one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptide(s)is determined using an antibody-based technique (e.g., immunoblotting oran enzyme-linked immunosorbent assay (ELISA)).

In all of the above aspects, a stress granule-related disorder may be acardiovascular disorder (e.g., an aneurysm, angina, atherosclerosis,stroke, cerebrovascular disease, congestive heart failure, coronaryartery disease, myocardial disease, peripheral vascular disease,granulomatous myocarditis, chronic myocarditis, myocardial infarction,and primary hypertrophic cardiomyopathy), an inflammatory disorder(e.g., an autoimmune disease, asthma, an allergic intraocularinflammatory disease, arthritis, atopic dermatitis, atopic eczema,cirrhosis, Crohn's disease, ulcerative colitis, diabetes, hemolyticanemia, inflammatory dermatosis, an inflammatory bowel disorder,systemic lupus erythamatosus, psoriasis, rheumatoid arthritis, Wegener'sgranulomatosis, Hashimoto's thyroiditis, chronic pancreatitis, andreactive lymphoid hyperplasia), a neurological disorder (e.g., multiplesclerosis, Alzheimer's disease, Parkinson's disease, Huntingon'sdisease, amyotrophic lateral sclerosis, retinosa pigmentosum, maculardegeneration, traumatic brain injury, stroke, and peripheralneuropathy), or ischemic-reperfusion injury. In different embodiments ofall the above aspects, the treating may result in a reduction (e.g., atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%reduction) in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)symptoms of a stress granule-related disorder or a reduction (e.g., atleast a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%reduction) in the likelihood of developing a stress granule-relateddisorder.

In all the above aspects, the cancer may be colon adenocarcinoma,esophagas adenocarcinoma, liver hepatocellular carcinoma, squamous cellcarcinoma, pancreas adenocarcinoma, islet cell tumor, rectumadenocarcinoma, gastrointestinal stromal tumor, stomach adenocarcinoma,adrenal cortical carcinoma, follicular carcinoma, papillary carcinoma,breast cancer, ductal carcinoma, lobular carcinoma, intraductalcarcinoma, mucinous carcinoma, phyllodes tumor, Ewing's sarcoma, ovarianadenocarcinoma, endometrium adenocarcinoma, granulose cell tumor,mucinous cystadenocarcinoma, cervix adenocarcinoma, vulva squamous cellcarcinoma, basal cell carcinoma, prostate adenocarcinoma, giant celltumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma,kidney carcinoma, urinary bladder carcinoma, Wilm's tumor, lymphoma, andnon-Hodgkin's lymphoma. In different embodiments of all the aboveaspects, said treating may result in a reduction (e.g., at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% reduction) inone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) symptoms of acancer or a reduction (e.g., at least a 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or even 100% reduction) in the likelihood ofdeveloping a cancer.

In all the above aspects of the invention, the cell or population ofcells is an epithelial cell, a fibroblast, a kidney cell, a muscle cell,a neuron, a hepatocyte, an oocyte, a sperm, a lymphocyte, or amacrophage.

In all the above aspects of the invention, the compositions may beformulated for parenteral or oral administration.

By the term “ARH3” or “poly-ADP-ribose glycohydrolase ARH3” is meant anucleic acid having containing a sequence at least 80% identical (e.g.,at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) tothe sequence of SEQ ID NO: 41, or one or more polypeptides encoded bythese nucleic acids.

By the term “ARH3 fusion protein” or “poly-ADP-ribose glycohydrolaseARH3 fusion protein” is meant a polypeptide containing a polypeptide tagand a sequence having at least 80% identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100% identity) to a protein encoded byARH3 (SEQ ID NO: 41). The polypeptide tag of an ARH3 fusion protein maybe located at the N- and/or C-terminus of the protein. The polypeptidetag may contain one or more of a fluorescent protein (e.g., a greenfluorescence protein), a peptide epitope recognized by specificantibodies, a protein that is bound by a partner binding protein withhigh affinity (e.g., biotin and streptavidin), a His₆-tag, or one ormore (e.g., 1, 2, 3, 4, 5, 6, or 7) protease recognition sequence(s)(e.g., one or more of a TEV protease or Factor Xa protease recognitionsequence). ARH3 fusion proteins may be purified using antibodiesspecific for the polypeptide tag. For example, antibodies specific forthe polypeptide tag or proteins that bind specifically to the proteinsequence in the polypeptide tag may bound to a bead (e.g., a magneticbead) or polymer surface in order to allow for the purification of theARH3 fusion protein. An ARH3 fusion protein may also be purified andsubsequently treated with one or more (e.g., 1, 2, or 3) protease(s) toremove the polypeptide tag from the ARH3 fusion protein. An ARH3 fusionprotein preferably has the same cellular localization and biologicalactivity as the wild-type ARH3 protein. Methods for the generation andpurification of ARH3 fusion proteins are described herein.

By the term “biotinylated” is meant the covalent attachment of a biotinmolecule to a small molecule, surface, or protein. A biotin molecule maybe attached to a small molecule, surface, or protein using methods knownin the art including, but not limited to, attachment to primary amines(e.g., epsilon-amines and N-terminal α-amines of a protein), as well asattachment at a sulfhydryl group, and a carboxyl group. Small molecules(e.g., NAD⁺) and proteins (e.g., one or more of the PARP fusion proteinsdescribed herein) may be biotinylated. Biotinylated NAD⁺ is availablefrom a number of commercial sources including R & D Systems, Gentaur,and Trevigen (e.g., 6-biotin-17-NAD). Biotinylated small molecules andsubstrates may be specifically bound and/or purified using streptavidin,a protein that has a high affinity for biotin (Ka˜10¹³ M⁻¹), or surfacescovalently attached to streptavidin (e.g., streptavidin-coated beads).

By the term “cancer” is meant a disease of uncontrolled or misregulatedcell proliferation or cell division. Non-limiting examples of cancerinclude colon adenocarcinoma, esophagas adenocarcinoma, liverhepatocellular carcinoma, squamous cell carcinoma, pancreasadenocarcinoma, islet cell tumor, rectum adenocarcinoma,gastrointestinal stromal tumor, stomach adenocarcinoma, adrenal corticalcarcinoma, follicular carcinoma, papillary carcinoma, breast cancer,ductal carcinoma, lobular carcinoma, intraductal carcinoma, mucinouscarcinoma, phyllodes tumor, Ewing's sarcoma, ovarian adenocarcinoma,endometrium adenocarcinoma, granulose cell tumor, mucinouscystadenocarcinoma, cervix adenocarcinoma, vulva squamous cellcarcinoma, basal cell carcinoma, prostate adenocarcinoma, giant celltumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma,kidney carcinoma, urinary bladder carcinoma, Wilm's tumor, lymphoma, andnon-Hodgkin's lymphoma.

By the term “cell lysate” is meant the contents of the cell once theplasma membrane has been disrupted or permeabilized. Cell lysate alsoincludes the contents of the intracellular organelles (e.g., endoplasmicreticulum, nucleus, mitochondria, chloroplasts, Golgi apparatus, andlysosome) upon disruption of their respective membranes. Cell lysatecontains an unpurified mixture of proteins, small molecule metabolites,and nucleic acids (e.g., DNA and RNA). Cell lysate may be prepared fromany type of cell, e.g., a mammalian cell (e.g. human, mouse, rat, andmonkey cell), a bacterial cell, fungal cell, and a yeast cell. Celllysate may be obtained by any methods known in the art includingphysical disruption (e.g., sonication, homogenization, or freeze/thawprocedures) or chemical disruption (e.g., treatment with a detergent(e.g., Triton-X-100 and NP-40)). Cell lysate may be prepared from a cellexpressing one or more of the nucleic acid(s) that encode one or morePARP, PARG, and/or ARH3 proteins and/or one or more PARP, PARG, and/orARH3 fusion protein(s). Cell lysate may also be prepared from a cellarrested in a specific stage of the cell cycle (e.g., mitosis orS-phase) or may be prepared from asynchronous cells.

By the term “constitutive promoter” is meant a promoter that is placed5′ relative to a nucleic acid sequence encoding a protein, wherein thepromoter regulates the consistent expression of a nucleic acid encodinga protein. The sequence of the constitutive promoter may be directly (noextraneous nucleotides) 5′ to the first nucleotide of the sequenceencoding the protein (e.g., a PARP, PARG, and/or ARH3 protein and/or aPARP, PARG, and/or ARH3 fusion protein as described herein) or may bebetween 1-20 nucleotides, 1-100 nucleotides, 10-260 nucleotides, 100-700nucleotides, or 100 to 2,000 nucleotides from the first nucleotide ofthe sequence encoding the protein. Examples of constitute promotersinclude, but are not limited to, bacterial promoters (e.g., E. coli σ⁷⁰, σ^(S), σ³², or σ⁵⁴ promoters; B. subtilis σ ^(A) or σ^(B) promoters;T7 RNA polymerase-based promoters; and bacteriophage SP6 promoter),yeast promoters (e.g., pCyc, pAdh, pSte5, ADH1, cyc100, cyc70, cyc43,cyc28, cyc16, pPGK1, pCYC, GPD (TDH3), and CLB1 promoters), andmammalian promoters (e.g., cytomegalovirus immediate early gene-basedpromoters, SV40 early promoter, and Rous sarcoma virus promoter). Aconstitutive promoter may be used to mediate the expression of a nucleicacid (e.g., one or more nucleic acids encoding a PARP, PARG, and/or ARH3protein, and/or a PARP, PARG, and/or ARH3 fusion protein as describedherein) in a transgenic mammalian, bacterial, or yeast cell.

By the term “disassembly” of a stress granule is meant the process ofdeconstructing or dissolution of one or more stress granules in a cellor cell population. The disassembly of a stress granule may take placeby the catalytic activity of one or more proteins (e.g., one or more ofa PARG, ARH3, and/or PARP11, and/or a PARG, ARH3, and/or PARP11 fusionprotein). The disassembly of a stress granule in a cell or cellpopulation may occur following the removal of a stress condition (e.g.,washout of sodium arsenite or pateamine A from the culture medium).

By the term “effective amount” or “therapeutically effective amount” ismeant the amount of the agent administered to a subject, to a cell, orto a cell population that elicits a specific desirable effect. Forexample, the amount of an agent (e.g., one or more PARP inhibitor(s),one or more PARG activator(s), and/or one or more PARP11 activator(s))that decreases the number (e.g., prevents 1, 2, 3, 4, or 5 symptoms fromoccurring) or severity (e.g., decrease of at least 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in the severity) of oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) symptoms of a stressgranule-related disorder or the amount of an agent (e.g., one or morePARP inhibitor(s)) that decreases the number or severity of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) symptoms of cancer. Theeffective amount or therapeutically effective amount of an agent maydetermined by a skilled artisan using methods known in the art and themethods described herein.

By “formation” of a stress granule is meant a series of events that leadto the appearance of distinct protein- and RNA-containing stressgranules in the cytoplasm. The formation of a stress granule in a cellmay be accelerated by exposure to stress conditions, including, but notlimited to, chemical stress (e.g., sodium arsenite and pateamine A). By“nucleation” of a stress granule is meant one of the initial steps orinitial rate-limiting steps in the formation of a stress granule in acell. Examples of proteins involved in the formation or nucleation ofstress granules include, without limitation, PARP 5A, PARP12, PARP13.1,PARP13.2, and PARP 15.

By “labeled nicotinamide adenine dinucleotide” or “labeled NAD⁺” ismeant a molecule of nicotinamide adenine dinucleotide (NAD⁺) that iscovalently labeled with a fluorescent molecule, a colorimetric molecule,or a molecule that is recognized by a specific partner protein (e.g.,biotinylation), or labeled with a radioisotope. One example of a labeledNAD⁺ is biotinylated NAD⁺ (e.g., 6-biotin-14-NAD). Examples ofradiolabeled NAD⁺ include, but are not limited to, ¹⁴C-adenine-NAD⁺,³²P-NAD⁺, and ³H-NAD⁺. Additional examples of labeled NAD⁺ are known inthe art.

By the term “short RNA or DNA aptamer” is meant a short sequence of DNAor RNA nucleotides that binds to a specific target molecule (e.g., aprotein or a target RNA or DNA molecule). A DNA or RNA aptamer thatspecifically binds to its target molecule (e.g., one or more (e.g., 1,2, 3, 4, or 5) of the nucleic acids encoding a PARP, PARG, and/or ARH3protein, and/or a PARP, PARG, and/or ARH3 fusion protein (as describedherein) may decrease (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100%) or increase (e.g., by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100%) one or more (e.g., 1, 2, 3, 4, or5) activities or expression (e.g., mRNA or protein level) of therespective target molecule. For example, a specific DNA or RNA aptamermay bind to one or more of the above-described PARP proteins or PARPfusion proteins and increase or decrease the poly-ADP ribosylationactivity of the protein, the amount of poly-ADP ribose attached to theprotein, or the levels of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) PARP proteins or PARP fusion proteins. The specific DNA or RNAaptamer may also bind to one or more nucleic acids (e.g., DNA or RNA)that encode a specific PARP, PARG, and/or ARH3 protein (e.g., a nucleicacid that encodes a protein having at least 80% sequence identity (e.g.,at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARP1 (SEQID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP3 (SEQ ID NO: 4), PARP3.2 (SEQID NO: 5), PARP3.3 (SEQ ID NO: 6), PARP4 (SEQ ID NO: 7), PARP5A (SEQ IDNO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP6 (SEQ ID NO: 11), PARP7 (SEQID NO: 12), PARP8 (SEQ ID NO: 13), PARP9 (SEQ ID NO: 14), PARP10 (SEQ IDNO: 15), PARP10.2 (SEQ ID NO: 16), PARP11 (SEQ ID NO: 17), PARP12 (SEQID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO: 20), PARP14(SEQ ID NO: 21), PARP15.1 (SEQ ID NO: 22), PARP15.2 (SEQ ID NO: 23),PARP16 (SEQ ID NO: 24), PARG (SEQ ID NO: 42), and ARH3 (SEQ ID NO: 41)),and mediate an increase or decrease in the expression (e.g., proteinand/or mRNA level) of the PARP, PARG, or ARH3. A specific example of anRNA aptamer is an inhibitory RNA (RNAi) molecule. Methods for the designof RNAi molecules are known in the art. Examples of specific RNAimolecules that may be used to decrease the expression of a PARP, PARG,and/or ARH3 protein, and/or PARP, PARG, and/or ARH3 fusion protein aredescribed herein.

By the term “fluorescent protein” is meant a protein that absorbs lightof a specific wavelength (e.g., absorption wavelength) and emits lightwith a longer wavelength (e.g., emission wavelength). The termfluorescent protein encompasses natural fluorescent proteins (i.e., thenatural form of the fluorescent protein without any geneticmanipulations) and genetically mutated fluorescent proteins (e.g.,fluorescent proteins engineered to change the identity of one or moreamino acid residues). Several different examples of fluorescent proteinsare known in the art, including, but limited to, green fluorescentproteins (e.g., GFP, Emerald, Superfolder GFP, Azami Green, mWasabi,TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and T-Sapphire), bluefluorescent proteins (e.g., EBFP, EBFP2, Azurite, mTagBFP), cyanfluorescent proteins (e.g., ECFP, mECFP, Cerulean, CyPet, AmCyan1,Midori-Ishi Cyan, TagCFP, mTFP1 (Teal)), yellow fluorescent proteins(e.g., EYFP, Topaz, Venus, mCitrine, YPet, TanYFP, PhiYFP, ZsYellow1,and mBanana), orange fluorescent proteins (e.g., Kurabira Orange,Kurabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP,TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, andmTangerine), and red fluorescent proteins (e.g., mRuby, mApple,mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry,dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143). Fluorescent proteins maybe attached to the N- and/or C-terminus of a target protein (e.g., oneor more of the PARP, PARG, and/or ARH3 fusion proteins describedherein). Fusion proteins tagged with a fluorescent protein (e.g., one ormore of the PARP, PARG, and/or ARH3 fusion proteins described herein)may be analyzed using fluorescence-based techniques known in the art(e.g., fluorescence microscopy, fluorescence plate readers,fluorescence-assisted cell sorting, and use of a second antibodyspecific for the fluorescent protein).

By the term “inducible promoter” is meant a promoter that is placed 5′relative to a nucleic acid sequence encoding a protein, wherein thepromoter induces (or represses) the expression of a nucleic acid uponaddition (or removal) of a specific molecule or protein. The sequence ofthe inducible promoter may be directly (no extraneous nucleotides) 5′ tothe first nucleotide of the sequence encoding the protein (e.g., a PARPfusion protein as described herein) or may be between 1-20 nucleotides,1-100 nucleotides, 10-260 nucleotides, 100-700 nucleotides, or 100 to2,000 nucleotides from the first nucleotide of the sequence encoding theprotein. Examples of inducible promoters include, but are not limited toalcohol dehydrogenase I gene promoters, tetracycline-responsive promotersystems, glucocorticoid receptor promoters, estrogen receptor promoter,ecdysone receptor promoters, metallothionein-based promoters, andT7-polymerase based promoters. An inducible promoter may be used toregulate the expression of a nucleic acid (e.g., one or more nucleicacids encoding a PARP, PARG, and/or ARH3 protein and/or PARP, PARG,and/or ARH3 fusion protein as described herein) in a transgenicmammalian, bacterial, or yeast cell.

By the term “nuclear lysate” is meant the contents of a nucleus upondisruption of the nuclear membrane. Nuclear lysate contains anunpurified mixture of proteins, small molecule metabolites, and nucleicacids (e.g., DNA and RNA). Nuclear lysate may be prepared from any typeof nucleated cell, e.g., a mammalian cell (e.g. human, mouse, rat, andmonkey cell), a fungal cell, a yeast cell, or a plant cell. Nuclearlysate may be obtained by any methods known in the art including steppedlysis using two different concentrations of detergents (e.g., NP-40) ora combination of physical treatment to rupture the plasma membrane andchemical treatment to rupture the nuclear membrane. Nuclear lysate maybe prepared from a cell expressing one or more of the nucleic acid(s) ofthe invention that encode a one or more PARP, PARG, or ARH3 proteins orPARP, PARG, or ARH3 fusion protein(s).

By “PAR” or “poly-ADP ribose” is meant a chain of two or more ADP-ribosemolecules. The two or more molecules of ADP-ribose making up PAR mayoccur in a single linear chain or as a branched chain with one or morebranches (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 branches).Poly-ADP ribose may be attached to a specific substrate (e.g., protein,lipid, DNA, RNA, or small molecule) by the activity of one or more PARPproteins or PARP fusion proteins (e.g., one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) ofPARP1, PARP2, PARP3, PARP3.2, PARP3.3, PARP4, PARP5A, PARP5B, PARP6,PARP7, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13.1, PARP13.2, PARP14,PARP15.1, PARP15.2, and PARP16, or one or more of their respectivefusion proteins) or removed by the activity of one or more PARG protein,PARG fusion protein, ARH3 protein, or ARH3 fusion protein (e.g., PARGprotein or ARH3). Attachment of poly-ADP-ribose to a substrate proteinmay affect the biological activity of the substrate protein,localization of the protein, or the identity and number of proteins thatbind to the target substrate (e.g., protein). PARP proteins may also bemodified by the covalent attachment of poly-ADP-ribose. The addition ofpoly-ADP ribose to a PARP protein may occur by “auto-modification” or“auto-modulation” (i.e., a specific PARP catalyzes the attachment ofpoly-ADP ribose to itself) or may occur by the activity of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) other PARP proteins.

By the term “poly-ADP-ribose glycolase” or “PARG” is meant any enzymethat has the ability to remove an ADP-ribose attached to a substrate(e.g., a protein, RNA molecule, DNA molecule, or lipid) or to remove oneor more ADP-ribose molecules from a pre-existing poly-ADP-ribosemolecule covalently attached to a substrate (e.g., a protein, RNAmolecule, DNA molecule, or lipid). For example, a PARG may be one ormore nucleic acids containing a sequence having at least 80% identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARG(SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41), or one or more polypeptidesencoded by these nucleic acids. A PARG may have additional biologicalactivities, such as decreasing (e.g., by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or even 100%) the formation or rate offormation of a stress granule in a cell or increasing the rate ofdisassembly of a stress granule. The term PARG also includes theisoforms of PARG proteins described in Meyer-Ficca et al., Exp. Cell.Res. 297(2):521-532, 2004.

By the term “PARG protein” or “poly-ADP-ribose glycolase protein” ismeant is meant a polypeptide encoded by a nucleic acid containing asequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100% identical) to the sequence of SEQID NO: 42.

By the term “PARG fusion protein” or “poly-ADP-ribose glycolase fusionprotein” is meant a polypeptide containing a polypeptide tag and asequence encoded by a nucleic acid containing a sequence having at least80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%,or even 100% identity) to PARG (SEQ ID NO: 42). The polypeptide tag of aPARG fusion protein may be located at the N- and/or C-terminus of theprotein. The polypeptide tag may contain one or more of a fluorescentprotein (e.g., a green fluorescence protein), a peptide epitoperecognized by specific antibodies, a protein that is bound by a partnerbinding protein with high affinity (e.g., biotin and streptavidin), aHis₆-tag, or one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) proteaserecognition sequence(s) (e.g., one or more of a TEV protease or FactorXa protease recognition sequence). PARG fusion proteins may be purifiedusing antibodies specific for the polypeptide tag. For example,antibodies specific for the polypeptide tag or proteins that bindspecifically to the protein sequence in the polypeptide tag may bound toa bead (e.g., a magnetic bead) or polymer surface in order to allow forthe purification of the PARG fusion protein. A PARG fusion protein mayalso be purified and subsequently treated with one or more (e.g., 1, 2,or 3) protease(s) to remove the polypeptide tag from the PARG fusionprotein. A PARG fusion protein preferably has the same cellularlocalization and biological activity as the wild-type PARG protein.Methods for the generation and purification of PARG fusion proteins aredescribed herein.

By the term “poly-ADP-ribose glycolase activator” or “PARG activator” ismeant an agent that increases (e.g., at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or even 100%) the expression (e.g., protein and/ormRNA level) or one or more (e.g., 1, 2, 3, 4, or 5) biologicalactivities of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARGproteins. For example, a PARG activator may increase the levels of oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acidscontaining a nucleic acid sequence having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARG(SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41), or increase the level of one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides encoded bythese nucleic acids. A PARG activator may increase one or more of thebiological activities of a PARG including the ability to remove aADP-ribose attached to a substrate (e.g., a protein, RNA molecule, DNAmolecule, lipid, or small molecule), the ability to remove one or moreADP-ribose molecules from a pre-existing poly-ADP-ribose moleculecovalently attached to one or more substrate(s) (e.g., a protein, RNAmolecule, DNA molecule, lipid, or small molecule), the ability todecrease or prevent the formation or the rate of formation of a stressgranule in a cell, or the ability to increase the rate of disassembly ofa stress granule. Non-limiting examples of PARG activators include oneor more nucleic acids containing a nucleic acid having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100%) to PARG (SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41).

By the term “poly-ADP-ribose glycolase inhibitor” or “PARG inhibitor” ismeant an agent that decreases (e.g., at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or even 100%) the expression (e.g., protein and/ormRNA level) or one or more (e.g., 1, 2, 3, 4, or 5) biologicalactivities of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)PARGs. For example, a PARG inhibitor may decrease the levels of one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acids containing anucleic acid sequence having at least 80% sequence identity (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARG (SEQ IDNO: 42) or ARH3 (SEQ ID NO: 41), or decrease the level of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides encoded by thesenucleic acids. A PARG activator may decrease one or more (e.g., 1, 2, 3,4, or 5) of the biological activities of a PARG including, but notlimited to, the ability to remove a ADP-ribose attached to one or moresubstrate(s) (e.g., a protein, RNA molecule, DNA molecule, lipid, orsmall molecule), the ability to remove one or more ADP-ribose moleculesfrom a pre-existing poly-ADP-ribose molecule covalently attached to asubstrate (e.g., a protein, RNA molecule, DNA molecule, lipid, or smallmolecule), the ability to decrease or prevent the formation or the rateof formation of a stress granule in a cell, or the ability to increasethe rate of disassembly of a stress granule. Non-limiting examples ofPARG inhibitors include antibodies or antibody fragments thatspecifically bind to PARG protein, ARH3 protein, PARG fusion protein, orARH3 fusion protein; RNAi molecules (e.g., a nucleic acid sequence thatcontains the sequence of one of SEQ ID NOS: 34-37), or small molecules.

By the term “peptide fragment” is meant a protein having at least 2amino acids (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids), but having feweramino acids than the wild-type protein. Non-limiting examples of peptidefragments have between 2 to 250 amino acids, 5 to 200 amino acids,between 5 to 150 amino acids, or between 5 to 100 amino acids. A peptidefragment may also represent a protein that has been processed to removeone or more (e.g., 1, 2, or 3) post-translational targeting sequences(e.g., nuclear localization sequence, ER signal peptide, mitochondrialtargeting signal, nuclear export sequence, or N-terminal secretionsequence).

By “poly-ADP ribose polymerase nucleic acid” or “PARP nucleic acid” ismeant any nucleic acid containing a sequence that has at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100% sequence identity) to one or more of PARP1 (SEQ ID NO: 1 or2), PARP2 (SEQ ID NO: 3), PARP3 (SEQ ID NO: 4), PARP3.2 (SEQ ID NO: 5),PARP3.3 (SEQ ID NO: 6), PARP4 (SEQ ID NO: 7), PARP5A (SEQ ID NO: 8 or9), PARP5B (SEQ ID NO: 10), PARP6 (SEQ ID NO: 11), PARP7 (SEQ ID NO:12), PARP8 (SEQ ID NO: 13), PARP9 (SEQ ID NO: 14), PARP10 (SEQ ID NO:15), PARP10.2 (SEQ ID NO: 16), PARP11 (SEQ ID NO: 17), PARP12 (SEQ IDNO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO: 20), PARP14 (SEQID NO: 21), PARP15.1 (SEQ ID NO: 22), PARP15.2 (SEQ ID NO: 23), andPARP16 (SEQ ID NO: 24). A PARP nucleic acid encodes a protein that has acatalytic activity of attaching an ADP-ribose to a substrate (e.g.,protein, DNA, RNA, lipid, or small molecule) or attaching one or moreADP-ribose molecules to an ADP-ribose molecule already attached to thesubstrate (e.g., protein, DNA, RNA, lipid, or small molecule) to createpoly-ADP ribose. A PARP nucleic acid may encode a protein havingadditional activities to those described above (e.g., mediates increasedstress granule formation, role in progression through mitosis orcytokinesis, and modulation (e.g., increase or decrease) of RNAifunction).

By “poly-ADP ribose polymerase protein” or “PARP protein” is meantpolypeptide containing a sequence having at least 80% identity (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% identity) to aprotein encoded by a nucleic acid sequence containing the sequence ofPARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP3 (SEQ ID NO: 4),PARP3.2 (SEQ ID NO: 5), PARP3.3 (SEQ ID NO: 6), PARP4 (SEQ ID NO: 7),PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP6 (SEQ ID NO:11), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP9 (SEQ ID NO:14), PARP10 (SEQ ID NO: 15), PARP10.2 (SEQ ID NO: 16), PARP11 (SEQ IDNO: 17), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQID NO: 20), PARP14 (SEQ ID NO: 21), PARP15.1 (SEQ ID NO: 22), PARP15.2(SEQ ID NO: 23), and PARP16 (SEQ ID NO: 24). A PARP protein may containone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) post-translationalmodifications, e.g., phosphorylation and ADP-ribosylation (e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ADP-ribose molecules) on one ormore amino acid residues. Post-translation modification of a PARPprotein may occur within a cell (e.g., a transgenic cell describedabove) or in vitro using purified enzymes. PARP protein activity assaysmay be performed as described herein.

By “poly-ADP ribose polymerase fusion protein” or “PARP fusion protein”is meant a polypeptide containing a polypeptide tag and a sequencehaving at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or even 100% identity) to a protein encoded by one or more ofPARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP3 (SEQ ID NO: 4),PARP3.2 (SEQ ID NO: 5), PARP3.3 (SEQ ID NO: 6), PARP4 (SEQ ID NO: 7),PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP6 (SEQ ID NO:11), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP9 (SEQ ID NO:14), PARP10 (SEQ ID NO: 15), PARP10.2 (SEQ ID NO: 16), PARP11 (SEQ IDNO: 17), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQID NO: 20), PARP14 (SEQ ID NO: 21), PARP15.1 (SEQ ID NO: 22), PARP15.2(SEQ ID NO: 23), and PARP16 (SEQ ID NO: 24). The polypeptide tag of aPARP fusion protein may be located at the N- and/or C-terminus of theprotein. The polypeptide tag may contain one or more of a fluorescentprotein (e.g., a green fluorescence protein), a peptide epitoperecognized by specific antibodies, a protein that is bound by a partnerbinding protein with high affinity (e.g., biotin and streptavidin), aHis₆-tag, or one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) proteaserecognition sequence(s) (e.g., one or more of a TEV protease or FactorXa protease recognition sequence). The PARP fusion proteins of theinvention may be purified using antibodies specific for the polypeptidetag. For example, antibodies specific for the polypeptide tag orproteins that bind specifically to the protein sequence in thepolypeptide tag may be bound to a bead (e.g., a magnetic bead) orpolymer surface in order to allow for the purification of the PARPfusion protein. A PARP fusion protein may also be purified andsubsequently treated with one or more (e.g., 1, 2, or 3) protease(s) toremove the polypeptide tag from the PARP fusion protein. A PARP fusionprotein preferably has the same cellular localization and biologicalactivity as the wild-type PARP protein. Methods for the generation andpurification of PARP fusion proteins are described herein.

By “PARP activator” or “poly-ADP-ribose polymerase activator” is meantan agent that increases the expression (e.g., mRNA or protein level)and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) biologicalactivities of one or more PARPs. For example, a PARP activator mayincrease the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) PARP nucleic acids or PARPproteins (described above). A PARP activator may increase one or morebiological activities of a PARP protein including, but not limited to,the ability to attach a poly-ADP-ribose molecule to one or moresubstrate(s) (e.g., a protein, DNA molecule, RNA molecule, lipid, orsmall molecule), the ability to promote formation of a stress granule,the ability to nucleate the formation of a stress granule, the abilityto disassemble a stress granule, the ability to decrease stress granuleassembly, the ability to localize to a stress granule, the ability of aPARP protein to bind to one or more of its substrates, the ability of aPARP protein to localize to the nucleus or the mitotic spindle, theability to promote cell proliferation, and the ability to promoteprogression through cytokinesis. Specific PARP activators includenucleic acids encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) PARPs or the catalyticdomains of one or more PARPs. For example, a PARP activator may be anucleic acid containing a nucleic acid sequence having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100%) to PARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP3(SEQ ID NO: 4), PARP3.2 (SEQ ID NO: 5), PARP3.3 (SEQ ID NO: 6), PARP4(SEQ ID NO: 7), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10),PARP6 (SEQ ID NO: 11), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13),PARP9 (SEQ ID NO: 14), PARP10 (SEQ ID NO: 15), PARP10.2 (SEQ ID NO: 16),PARP11 (SEQ ID NO: 17), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO:19), PARP13.2 (SEQ ID NO: 20), PARP14 (SEQ ID NO: 21), PARP15.1 (SEQ IDNO: 22), PARP15.2 (SEQ ID NO: 23), and PARP16 (SEQ ID NO: 24). SpecificPARP activators may increase the expression and/or one or more (e.g., 1,2, 3, 4, or 5) biological activities of a specific PARP or a specificsubset of PARPs (e.g., one or more of PARP5A, PARP12, PARP13.1,PARP13.2, and PARP15; PARP11; and one or more of PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16).

By “PARP inhibitor” or “poly-ADP-ribose polymerase inhibitor” is meantan agent that decreases the expression (e.g., mRNA or protein level)and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) biologicalactivities of one or more PARPs. For example, a PARP inhibitor maydecrease the level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) PARP nucleic acids or PARPproteins (described above). A PARP inhibitor may decrease one or more(e.g., 1, 2, 3, 4, or 5) biological activities of a PARP proteinincluding, but not limited to, the ability to attach a poly-ADP-ribosemolecule to a substrate (e.g., a protein, DNA molecule, RNA molecule,lipid, or small molecule), the ability to promote formation of a stressgranule, the ability to nucleate the formation of a stress granule, theability to disassemble a stress granule, the ability to decrease stressgranule assembly, the ability to localize to a stress granule, theability of a PARP protein to bind to one or more of its substrates, theability of a PARP protein to localize to the nucleus or the mitoticspindle, the ability to promote cell proliferation, and the ability topromote progression through cytokinesis. Specific PARP inhibitorsinclude antibody or antibody fragments that specifically bind one ormore PARP proteins (described herein), one or more RNA aptamers (e.g.,RNAi molecules; e.g., SEQ ID NOS: 40 and 43-136), and one or more smallmolecules. Specific PARP inhibitors may decrease the expression and/orone or more biological activities (e.g., 1, 2, 3, 4, or 5) of a specificPARP or a specific subset of PARPs (e.g., one or more (e.g., 1, 2, 3, 4,or 5) of PARP5A, PARP12, PARP13.1, PARP13.2, and PARP15; PARP11; and oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16).

By “Poly-ADP-ribose polymerase 11 activator” is meant an agent thatincreases the expression (e.g., mRNA or protein level) and/or one ormore (e.g., 1, 2, 3, 4, or 5) biological activities of PARP11. Forexample, a PARP11 activator may increase the level of one or more (e.g.,1, 2, 3, 4, or 5) nucleic acids containing a sequence having at least80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%,or even 100%) to PARP11 (SEQ ID NO: 17), and/or increase the level ofone or more (e.g., 1, 2, 3, 4, or 5) polypeptides encoded by thesenucleic acids. A PARP11 activator may increase one or more (e.g., 1, 2,3, 4, or 5) biological activities of a PARP11 protein including, but notlimited to, the ability to attach a poly-ADP-ribose molecule to asubstrate (e.g., a protein, DNA molecule, RNA molecule, lipid, or smallmolecule), the ability to prevent or reduce the rate of formation of astress granule, the ability to prevent the nucleation of a stressgranule, the ability to disassemble a stress granule, the ability todecrease stress granule assembly, the ability to localize to a stressgranule, or the ability of PARP11 protein to bind to one or more of itssubstrates. Specific PARP11 activators include nucleic acids encodingPARP11 or the catalytic domain of PARP11. For example, a PARP activatormay be a nucleic acid containing a nucleic acid sequence having at least80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%,or even 100%) to PARP11 (SEQ ID NO: 17). Specific PARP11 activators mayincrease the expression and/or one or more biological activities of aPARP11, while having little or no effect on the expression and/or one ormore biological activities of other PARP proteins.

By “poly-ADP-ribose polymerase-11 inhibitor” or “PARP11 inhibitor” ismeant an agent that decreases the expression (e.g., mRNA or proteinlevel) and/or one or more (e.g., 1, 2, 3, 4, or 5) biological activitiesof PARP11. For example, a PARP11 inhibitor may decrease the level of oneor more (e.g., 1, 2, 3, 4, or 5) nucleic acids containing a sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or even 100%) to PARP11 (SEQ ID NO: 17), or decreasethe level of one or more (e.g., 1, 2, 3, 4, or 5) polypeptides encodedby these nucleic acids. A PARP11 inhibitor may decrease one or more(e.g., 1, 2, 3, 4, or 5) biological activities of a PARP11 proteinincluding, but not limited to, the ability to attach a poly-ADP-ribosemolecule to a substrate (e.g., a protein, DNA molecule, RNA molecule,lipid, or small molecule), the ability to prevent or reduce the rate offormation of a stress granule, the ability to prevent the nucleation ofa stress granule, the ability to disassemble a stress granule, theability to decrease stress granule assembly, the ability to localize toa stress granule, or the ability of PARP11 protein to bind to one ormore of its substrates. Specific PARP11 inhibitors include antibody orantibody fragments that specifically bind to PARP11 protein, one or moreRNA aptamers (e.g., RNAi molecules; e.g., SEQ ID NOS: 91-98), and one ormore small molecules. Specific PARP11 inhibitors decrease the expressionand/or one or more (e.g., 1, 2, 3, 4, or 5) biological activities ofPARP11, while having little or no effect on the expression and/oractivity of other PARP proteins.

By “PARP biological activity” is meant one or more (e.g., 1, 2, 3, 4, or5) of the ability of a PARP protein or PARP fusion protein to catalyzethe attachment of a single ADP-ribose to a target substrate (e.g., aprotein, DNA, RNA, lipid, or small molecule), the ability to attach oneor more ADP-ribose molecules to a ADP-ribose molecule already attachedto a substrate, the ability to add a branched ADP-ribose molecule to apre-existing poly-ADP-ribose, the ability to localize to the cellnucleus, the ability to localize to stress granules, the ability tocatalyze the formation or nucleate stress granules, the ability tocatalyze the disassembly of stress granules, the ability to promote celldivision or progression through mitosis, or the ability to activate orinhibit RNAi activity in the cell. Specific PARP proteins have adifferent subset of biological activities. For example, PARP1, PARP2,PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16 have the ability tolocalize to the nucleus and play a role in mitosis and cell division.PARP 5A, PARP12, PARP13.1, PARP13.2, and PARP-15 have the ability tolocalize to stress granules and play a role in the formation ornucleation of stress granules. PARP11 has the ability to localize tostress granules and plays a role in inhibiting stress granule formationor increasing the disassembly of stress granules. PARP13 inhibits theactivity of RNAi in the cell. An additional PARP activity is“auto-modification”or “auto-modulation,” that is, attachment of one ormore ADP-ribose molecules to itself. Such auto-modulation of a PARPprotein may result in an increase or decrease in any of the above-listedPARP activities. Assays for the measurement of the activity of eachspecific PARP are described herein.

By “polypeptide tag” is meant a protein sequence that is located at the5′ and/or 3′ end of a polypeptide sequence of an expressed protein(e.g., one or more PARP proteins as described herein). A polypeptide tagmay include one or more of a protease recognition sequence (e.g., 1, 2,3, 4, 5, or 6 of the same or different protease recognition sequences),a epitope tag (e.g., 1, 2, 3, 4, or 5 epitope tags), a peptide that hasa high affinity binding partner (e.g., biotin and streptavidin), or oneor more (e.g., 1, 2, 3, or 4) tag(s) which aids in protein purification(e.g., a His₆ tag). The polypeptide tag may later be cleaved from thepurified fusion protein by incubation with one or more (e.g., 1, 2, 3,or 4) protease(s) which cleaves the fusion protein at one or moreprotease recognition sequence(s) (e.g., 1, 2, 3, 4, 5, 6, or 7) withinthe sequence of the polypeptide tag. Examples of polypeptide tags aredescribed herein.

By “positioned 3′” is meant a second nucleic acid sequence that islocated after the 3′ terminus of a first nucleic acid sequence (thesecond nucleotide sequence starts at the nucleotide following the 3′terminus of the first sequence) or the second nucleic acid sequencebegins at a nucleotide that follows the 3′ terminus of the first nucleicacid (e.g., the second nucleotide sequence starts at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, or 400 nucleotides following the 3′ terminus of the firstnucleic acid).

By “positioned 5′” is meant a second nucleic acid sequence that islocated before the 5′ terminus of a first nucleic acid sequence (thesecond nucleotide sequence ends at the nucleotide preceding the 5′terminus of the first sequence) or the second nucleic acid sequence endsat a nucleotide that precedes the 5′ terminus of the first nucleic acid(e.g., the second nucleotide sequence ends at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300,or 400 nucleotides before the 5′ terminus of the first nucleic acid).

By the term “propensity to develop disease” is meant the calculatedprobability of a subject (e.g., a human) to develop a disease (e.g., astress granule-related disorder or cancer). The probability ofdeveloping a disease may be calculated based on a number of factorsincluding a variety of health indicators (e.g., blood pressure,cholesterol, and levels of pro-inflammatory cytokines), biologicalfactors (e.g., weight, age, and sex), and genetic susceptibility todisease (e.g., expression of a heritable mutation in a gene, expressionof a polymorphic sequence associated with a disease, and expression ofan allele associated with a disease). The propensity to develop diseasein a specific patient population may be compared to a different patientpopulation (e.g., a patient population not receiving a therapy).

By the term “protease recognition sequence” is meant a short peptidesequence that is recognized as a substrate and cleaved by one or more(e.g., 1, 2, 3, 4, or 5) proteases. Protease target sequences are often3-20 amino acids in length and often require certain amino acids to belocated at specific positions within the target sequence, while anyamino acid may be placed at other positions within the target sequence.For example, the protease recognition sequence for TEV protease isGlu-X-X-Tyr-X-Gln-Ser (SEQ ID NO: 26), where X represents a positionthat may be filled by any amino acid. Additional examples of proteaserecognition sequences are known in the art and include, withoutlimitation, factor Xa (Ile-Glu/Asp-Gly-Arg), Ala-64 subtilisin(Gly-Ala-His-Arg), clostripain (Arg and Lys-Arg), collagenase(Pro-Val-Gly-Pro), enterokinase (Asp-Asp-Asp-Asp-Lys), renin(Pro-Phe-His-Leu-Leu), and α-thrombin (Leu-Val-Pro-Arg-Gly-Ser). One ormore of the same or different protease recognition sequence(s) may beincluded in the polypeptide tag of any of the PARP, PARG, or ARH3 fusionproteins described herein. A protease recognition sequence may be placed5′ or 3′ to an amino acid sequence to be removed from the protein. Thepolypeptide sequence of the protease recognition sequence may directlyabut the sequence encoding a PARP or may be separated from the remainingcoding sequence by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50amino acids). An amino acid sequence that may be removed from theprotein may include one or more antigenic sequence(s), a His₆-tag, afluorescent protein, a peptide sequence that has high affinity to asecond protein that was used to purify the protein (e.g., His₆ tag orhemagglutinin tag), or a peptide sequence that was used to stabilize theprotein during purification (e.g., albumin).

By the term “purified” is meant purified from other common componentsnormally present within the cell. For example, a purified protein ispurified away from the other cellular proteins, nucleic acids, and smallmetabolites present within the cell. A purified protein is at least 85%pure by weight (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or even100% pure) from other proteins, nucleic acids, or small metabolitespresent in the cell. A purified nucleic acid is at least 85% free ofother contaminating nucleic acid molecules or adjoining sequences foundin the cell.

By the term “rate of proliferation” is meant the rate of a cell or acell population to undergo successive cell divisions. The rate of cellproliferation of a cell or cell population may be measured by methodsknown in the art including cell counting (e.g., by microscopictechniques) and the incorporation of labeled nucleotides into newlysynthesized DNA (e.g., incorporation of ³H-thymidine). The rate ofproliferation of a cell or cell population treated with one or moreagent(s) (e.g., one or more PARP activators or PARP inhibitors) may becompared to a control cell or cell population not treated with theagent.

By the term “reduce the likelihood of developing” is meant a reduction(e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%,80%, 90%, or 95%) for an individual or a patient population in thechance or rate of developing a specific disease by administering one ormore therapeutic agent(s) compared to an individual or patientpopulation not receiving the therapeutic agent. The methods of theinvention may also reduce the likelihood of developing one or more(e.g., 1, 2, 3, 4, or 5) symptoms of a stress granule-related disorderor reduce the likelihood of developing one or more (e.g., 1, 2, 3, 4, or5) symptoms of cancer in a patient population or an individual receivingone or more therapeutic agent(s).

By the term “RNAi” is meant a short double-stranded RNA molecule thatmediates the down-regulation of a target mRNA in a cell. An RNAimolecule is typically 15 to 32 nucleotides in length. RNAi molecules arealso known as siRNAs, small RNAs, or microRNAs. The design andtherapeutic effectiveness of RNAi molecules is described in McCaffrey etal. (Nature 418:38-39, 2002). The RNAi molecules are at least 15nucleotides, preferably, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, or 35 nucleotides in length and even up to 50 or100 nucleotides in length (inclusive of all integers in between).Non-limiting examples of RNAi molecules are at least 80% identical(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%identical) to or complementary to the translational start sequence orthe nucleic acid sequence encoding the first 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 amino acids of a PARP, PARG, or ARH3 selected from anucleic acid sequence containing a sequence at least 80% identical toone of PARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP3 (SEQ IDNO: 4), PARP3.2 (SEQ ID NO: 5), PARP3.3 (SEQ ID NO: 6), PARP4 (SEQ IDNO: 7), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP6 (SEQID NO: 11), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP9 (SEQ IDNO: 14), PARP10 (SEQ ID NO: 15), PARP10.2 (SEQ ID NO: 16), PARP11 (SEQID NO: 17), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2(SEQ ID NO: 20), PARP14 (SEQ ID NO: 21), PARP15.1 (SEQ ID NO: 22),PARP15.2 (SEQ ID NO: 23), PARP16 (SEQ ID NO: 24), PARG (SEQ ID NO: 42),or ARH3 (SEQ ID NO: 41). An RNAi molecule may target any part of thesequence encoding the target protein (e.g., any part of an mRNA encodingone of the above listed PARP, PARG, or ARH3 proteins).

The specific requirements and modifications of small RNA are known inthe art and are described, for example in PCT Publication No.WO01/75164, and U.S. Application Publication Nos. 20060134787,20050153918, 20050058982, 20050037988, and 20040203145, the relevantportions of which are herein incorporated by reference. siRNAs can alsobe synthesized or generated by processing longer double-stranded RNAs,for example, in the presence of the enzyme dicer under conditions inwhich the dsRNA is processed to RNA molecules of about 17 to about 26nucleotides. siRNAs can also be generated by expression of thecorresponding DNA fragment (e.g., a hairpin DNA construct). Generally,the siRNA has a characteristic 2- to 3-nucleotide 3′ overhanging ends,preferably these are (2′-deoxy)thymidine or uracil. The siRNAs typicallycomprise a 3′ hydroxyl group. Single-stranded siRNAs or blunt-endeddsRNA may also be used. In order to further enhance the stability of theRNA, the 3′ overhangs may be stabilized against degradation. Forexample, the RNA may be stabilized by including purine nucleotides, suchas adenosine or guanosine. Alternatively, substitution of pyrimidinenucleotides by modified analogs, e.g., substitution of uridine2-nucleotide overhangs by (2′-deoxy)thymidine is tolerated and does notaffect the efficiency of RNAi. The absence of a 2′ hydroxyl groupsignificantly enhances the nuclease resistance of the overhang in tissueculture medium.

siRNA molecules can also be obtained through a variety of protocolsincluding chemical synthesis or recombinant production using aDrosophila in vitro system. They can be commercially obtained fromcompanies such as Dharmacon Research Inc. or Xeragon Inc., or they canbe synthesized using commercially available kits such as the Silencer™siRNA Construction Kit from Ambion (catalog number 1620) or HiScribe™RNAi Transcription Kit from New England BioLabs (catalog number E2000S).

Alternatively siRNA can be prepared using standard procedures for invitro transcription of RNA and dsRNA annealing procedures such as thosedescribed in Elbashir et al. (Genes & Dev., 15:188-200, 2001), Girard etal. (Nature 442:199-202, 2006), Aravin et al. (Nature 442:203-207,2006), Grivna et al. (Genes Dev. 20:1709-1714, 2006), and Lau et al.(Science 313:305-306, 2006). siRNAs may also be obtained by incubationof dsRNA that corresponds to a sequence of the target gene in acell-free Drosophila lysate from syncytial blastoderm Drosophila embryosunder conditions in which the dsRNA is processed to generate siRNAs ofabout 21 to about 23 nucleotides, which are then isolated usingtechniques known to those of skill in the art. For example, gelelectrophoresis can be used to separate the 21-23 nt RNAs and the RNAscan then be eluted from the gel slices. In addition, chromatography(e.g., size exclusion chromatography), glycerol gradient centrifugation,and affinity purification with antibody can be used to isolate the smallRNAs.

Short hairpin RNAs (shRNAs), as described in Yu et al. (Proc. Natl.Acad. Sci. U.S.A. 99:6047-6052, 2002) or Paddison et al. (Genes & Dev.16:948-958, 2002), incorporated herein by reference, may also be used.shRNAs are designed such that both the sense and antisense strands areincluded within a single RNA molecule and connected by a loop ofnucleotides (3 or more). shRNAs can be synthesized and purified usingstandard in vitro T7 transcription synthesis as described above and inYu et al. (supra). shRNAs can also be subcloned into an expressionvector that has the mouse U6 promoter sequences which can then betransfected into cells and used for in vivo expression of the shRNA.

A variety of methods and reagents are available for transfection, orintroduction, of dsRNA into mammalian cells including but not limitedto: TransIT-TKO™ (Mirus, Cat. # MIR 2150), Transmessenger™ (Qiagen, Cat.#301525), Oligofectamine™ and Lipofectamine™ (Invitrogen, Cat. # MIR12252-011 and Cat. #13778-075), siPORT™ (Ambion, Cat. #1631), andDharmaFECT™ (Fisher Scientific, Cat. # T-2001-01). Agents are alsocommercially available for electroporation-based methods fortransfection of siRNA, such as siPORTer™ (Ambion Inc. Cat. #1629).Microinjection techniques can also be used. The small RNA can also betranscribed from an expression construct introduced into the cells,where the expression construct includes a coding sequence fortranscribing the small RNA operably-linked to one or moretranscriptional regulatory sequences. Where desired, plasmids, vectors,or viral vectors can also be used for the delivery of dsRNA or siRNA andsuch vectors are known in the art. Protocols for each transfectionreagent are available from the manufacturer. Additional methods areknown in the art and are described, for example in U.S. PatentApplication Publication No. 20060058255.

By the term “specifically binds” is meant a protein, nucleic acid (e.g.,DNA or RNA), or molecule that binds one or more target molecules (e.g.,polypeptides, DNA molecules, or RNA molecules) present in a cell, whilenot binding the majority of other proteins, DNA molecules, RNAmolecules, or small molecules present within a cell, cell lysate,extracellular medium, or biological sample. For example, an antibodyprovided by the invention may bind to a single PARP, PARG, or AREprotein, a PARP, PARG, or ARH3 fusion protein, or may bind more than one(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 or more) PARP, PARG, or ARH3 proteins and/or PARP, PARG, or ARH3fusion proteins in a cell, cell lysate, extracellular medium, orbiological sample.

By “substrate” or “solid surface” is meant a surface on which a moietyor protein is covalently attached which allows for the binding and/orpurification of a PARP, PARG, and/or ARH3 fusion protein. The PARP,PARG, and/or ARH3 fusion protein will bind to the substrate or solidsurface through its polypeptide tag. Moieties or peptides covalentlyattached to the substrate or solid surface include, but are not limitedto, monoclonal or polyclonal antibodies specific for an antigenicpeptide in the polypeptide tag (e.g., anti-GFP antibody binding to GFPin the polypeptide tag), specific metal complexes bound by a peptidelocated in the polypeptide tag (e.g., Ni⁺ binding to a His₆ polypeptidetag), or a specific binding protein for a peptide located in thepolypeptide tag (e.g., IgG binding to a ZZ-domain in the polypeptidetag). Examples of a substrate or solid surface include, but are notlimited to, a bead (e.g., a magnetic bead), a surface in a multi-wellplate, and beads in column (e.g., column chromatography). One or morePARP, PARG, and/or ARH3 protein(s) and/or PARP, PARG, and/or ARH3 fusionprotein(s) may be bound to a substrate or solid surface and eluted fromthe substrate or solid surface by contacting the substrate or solidsurface with an elution buffer (e.g., a high salt elution buffer), aligand that competes for binding to the substrate or solid surface, orcompetes for binding to the polypeptide tag (e.g., a non-bound antibodythat specifically binds to the protein in the polypeptide tag), or bytreating the bound fusion protein with a protease that recognizes theone or more (e.g., 1, 2, 3, 4, 5, 6, or 7) specific cleavage recognitionsequence(s) found in the polypeptide tag.

By the term “stress granule-related disorder” is meant any disorder thatis characterized or in part caused by the activity or formation ofstress granules in a specific type of cell or cell population.Non-limiting examples of stress granule-related disorders includecardiovascular disorders (e.g., an aneurysm, angina, atherosclerosis,stroke, cerebrovascular disease, congestive heart failure, coronaryartery disease, myocardial disease, peripheral vascular disease,granulomatous myocarditis, chronic myocarditis, myocardial infarction,and primary hypertrophic cardiomyopathy), inflammatory disorders (e.g.,autoimmune diseases, asthma, allergic intraocular inflammatory diseases,arthritis, atopic dermatitis, atopic eczema, cirrhosis, Crohn's disease,ulcerative colitis, diabetes, hemolytic anemia, inflammatory dermatosis,an inflammatory bowel disorder, systemic lupus erythamatosus, psoriasis,and rheumatoid arthritis, Wegener's granulomatosis, Hashimoto'sthyroiditis, chronic pancreatitis, and reactive lymphoid hyperplasia),neurological disorders (e.g., multiple sclerosis, Alzheimer's disease,Parkinson's disease, Huntingon's disease, amyotrophic lateral sclerosis,retinosa pigmentosum, macular degeneration, traumatic brain injury,stroke, and peripheral neuropathy), and ischemia-reperfusion injury(e.g., stroke). A stress granule-related disorder is typicallycharacterized by a disease etiology that involves oxidative stress orthe production of oxygen-based radicals in a tissue over the progressionof the disease. Methods for the diagnosis of several stressgranule-related disorders are known in the art.

By the term “symptoms of a stress-granule related disorder” is meant oneor more (e.g., 1, 2, 3, 4, or 5) of the physical manifestations of astress-granule related disorder. Non-limiting examples of symptoms of astress-granule related disorder include pain, swelling, inflammation,loss of cognition, loss of vision, loss of coordination, difficultybreathing, airway constriction, artery occlusion, diarrhea, elevatedblood glucose, increased levels of pro-inflammatory cytokines, increasedprotein aggregates or deposits, and increased cell death (e.g.,apoptosis or necrosis).

By the term “symptoms of cancer” is meant one or more (e.g., 1, 2, 3, 4,or 5) of the physical manifestations of cancer. Non-limiting examples ofsymptoms of cancer include blood in urine, pain or burning uponurination, cloudy urine, pain in bone, fractures in bones, fatigue,weight loss, repeated infections, nausea, vomiting, constipation,numbness in the legs, bruising, dizziness, drowsiness, abnormal eyemovements, changes in vision, changes in speech, headaches, thickeningof a tissue, rectal bleeding, abdominal cramps, loss of appetite, fever,enlarged lymphnodes, persistent cough, blood in sputum, lung congestion,itchy skin, lumps in skin, abdominal swelling, vaginal bleeding,jaundice, heartburn, indigestion, cell proliferation, and loss ofregulation of controlled cell death.

By the term “target protein” or “substrate protein” is meant a proteinthat is bound by one or more (e.g., 1, 2, 3, 4, or 5) PARP protein(s),PARG protein(s), ARH3 protein(s), PARP fusion protein(s), PARG fusionprotein(s), and/or ARH3 fusion protein(s); covalently modified byattachment of a ADP-ribose molecule by the activity of one or more(e.g., 1, 2, 3, 4, or 5) PARP protein(s) or PARP fusion protein(s); orcontains a poly-ADP-ribosyl group that is hydrolyzed by the activity ofone or more (e.g., 1, 2, 3, 4, or 5) PARG proteins, PARG fusionproteins, ARH3 proteins, or ARH3 fusion proteins. A target or substrateprotein may be co-localized in the nucleus or in a stress granule,and/or may localize to the mitotic spindle during cytokinesis. A targetprotein or substrate protein may localize to different structures ororganelles within a cell during different stages of the cell cycle(e.g., interphase, S-phase, prophase, metaphase, telephase, andanaphase) and may have an activity in the formation, nucleation, ordisassembly of stress granules, an activity in cell proliferation orprogression through cytokinesis, or an activity in the regulation ofmiRNA or RNAi activity. A target or substrate protein may be a PARP,PARG, or ARH3 protein (described herein).

By the term “transgenic cell” is a meant a cell expressing one or morenucleic acids introduced by recombinant DNA technology. For example, atransgenic cell may express a nucleic acid encoding one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) of thepresently described PARP, PARG, and/or ARH3 proteins and/or one or moreof the PARP, PARG, and/or ARH3 fusion proteins. A transgenic cell may bea mammalian cell (e.g., a mouse, rat, monkey, or human cell), abacterial cell, a fungal cell, a yeast cell, or a plant cell. Thetransgenic cell may express the introduced nucleic acids from aninducible promoter or a constitutive promoter. The transgenic cell mayalso be located within a transgenic animal or may be cultured in tissueculture. The introduced one or more nucleic acid(s) may be integrated inthe chromosome of a cell or may be expressed from a plasmid.

By “ZZ-domain” is meant a polypeptide sequence encoded by a nucleic acidhaving at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or even 100% identity) to the Staphylcoccus aureus protein Adomain encoded by SEQ ID NO: 27. The ZZ domain has the ability to bindto Fcγ (the constant region of IgG involved in effector functions) andFab (the Ig fragment responsible for antigen recognition). The specificstructure and binding properties of the ZZ-domain are described inGraille et al. (Proc. Natl. Acad. Sci. U.S.A. 97:5399-5404, 2000) andRoben et al. (J. Immunol. 154:6437-6445, 1995). Expression of theZZ-domain in the polypeptide tag allows for the purification of a fusionprotein (e.g., one or more PARP fusion proteins as described herein) bythe use of an Fc-containing protein (e.g., IgG).

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains drawings executed in color (FIGS. 2, 5,9-11, 16-18, and 20-22). Copies of this patent or patent applicationwith color drawings will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 is a picture of the chemical structure of nicotinamide adeninedinucleotide (NAD⁺) and poly-ADP ribose.

FIG. 2 is a set of micrographs showing the mitotic localization ofpoly-ADP ribose in HeLa cells during G2-M, prophase, prometaphase (P-M),metaphase, anaphase, and cytokinesis stages of the cell cycle usingfluorescence microscopy following staining with rabbit anti-PARantibodies labeled with Alexa 488 and X-rhodamine NHS esters.

FIG. 3 is a set of schematic diagrams showing the domain organization ofPARP1, PARP2, PARP3, PARP4, tankyrase 1 (PARP5A), tankyrase 2 (PARP5B),TiPARP (PARP7), PARP12, PARP13, PARP9, PARP14, PARP15, PARP10, PARP11,PARP6, PARP8, and PARP16.

FIG. 4 is a diagram of the pEGFP-C1 vector (SEQ ID NO: 28) (Invitrogen)showing the CMV promoter, the EGFP sequence, the multiple cloning site(MCS), and the SV40 poly A sequence. Also shown is the polylinkersequence (SEQ ID NO: 29).

FIG. 5 is an immunoblot showing the expression and relative size of thePARP-GFP fusion proteins of PARP1, PARP2, PARP3, PARP4, PARP5A, PARP5B,PARP6, PARP7, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14,PARP15, and PARP16 expressed in HeLa Kyoto cells transfected withpEGFP-C1 plasmids containing a nucleic acid encoding each respectivePARP-GFP fusion protein. The immunoblot was developed using a rabbitanti-GFP polyclonal antibody.

FIG. 6 is a set of micrographs showing the localization of differentPARP-GFP fusion proteins in asynchronous HeLa Kyoto cells transfectedwith a pEGFP-C1 plasmid containing a nucleic acid encoding a PARP-GFPprotein. The transfected cells were immunostained with rabbit anti-GFPpolyclonal antibody and fluorescently-labeled secondary Alexa Fluor 594or 488 antibody (Invitrogen), and visualized using fluorescencemicroscopy. The localization of PARP1-GFP, PARP2-GFP, PARP3-GFP,PARP7-GFP, PARP12-GFP, PARP13-GFP, PARP9-GFP, PARP14-GFP, PARP15-GFP,PARP6-GFP, PARP8-GFP, PARP16-GFP, PARP4-GFP, PARP10-GFP, PARP11-GFP,PARP5A-GFP, and PARP5B-GFP fusion proteins is shown.

FIG. 7 is two sets of micrographs showing the localization of differentPARP-GFP fusion proteins in asynchronous hTERT-RPE and HeLa Kyoto cellstransfected with a pEGFP-C1 plasmid containing a nucleic acid encoding aPARP-GFP protein. The transfected cells were immunostained with rabbitanti-GFP polyclonal antibody and fluorescently-labeled Alexa Fluor 594or 488 antibodies (Invitrogen), and visualized using fluorescencemicroscopy. The localization of PARP1-GFP, PARP2-GFP, PARP3-GFP,PARP4-GFP, PARP5A-GFP, PARP5B-GFP, PARP6-GFP, PARP7-GFP, PARP8-GFP,PARP9-GFP, PARP10-GFP, PARP11-GFP, PARP12-GFP, PARP13-GFP, PARP14-GFP,PARP15-GFP, and PARP16-GFP fusion proteins is shown for each cell type.

FIG. 8 is a set of micrographs from asynchronous HeLa Kyoto cellstransfected with a pEGFP-C1 plasmid containing a nucleic acid encoding aPARP-GFP protein following immunostaining with primary rabbit antibodiesraised against each specific PARP and fluorescently-labeled Alexa Fluor594 or 488 antibodies (Invitrogen). The localization of PARP1, PARP2,PARP3, PARP4, PARP5A, PARP5B, PARP6, PARP7, PARP8, PARP9, PARP10,PARP11, PARP12, PARP13.1, PARP13.2, PARP14, PARP15, and PARP16 is shown.

FIG. 9 is a set of micrographs showing the localization of each PARP-GFPfusion protein during S-phase and mitosis in transfected HeLa Kyotocells. In each experiment, HeLa Kyoto cells were transfected usingLipofectamine 2000 with a specific PARP-GFP expression vector (pEGFP-C1)and were arrested in mitosis or S-phase by treatment with 100 nMnocodazole or 5 μg/mL aphidicolin for 12 hours. The resulting treatedcells were immunostained with rabbit anti-GFP polyclonal antibodies andfluorescently-labeled Alexa Fluor 594 or 488 antibodies (Invitrogen),and visualized using fluorescence microscopy. S-phase-arrested cellswere also stained with EdU and DAPI, and mitosis-arrested cells werefurther stained with tubulin and DAPI.

FIG. 10 is a set of micrographs showing the localization ofoverexpressed PARP16-GFP in the endoplasmic reticulum of HeLa Kyotocells transfected with a pEGFP-C1 plasmid encoding a PARP16-GFP fusionprotein (middle panel) and the phenotype of HeLa Kyoto cells transfectedwith an RNAi targeting endogenous PARP16 (right panel). The left panelshows untransfected HeLa Kyoto cells stained with anti-calnexinantibodies, secondary fluorescently-labeled antibodies (Alexa Fluor 594or 488 antibodies (Invitrogen)), and DAPI. The middle panel thelocalization of PARP16-GFP and calnexin in HeLa Kyoto cells transfectedwith a pEGFP-C1 plasmid expressing a PARP16-GFP fusion protein followingstaining with anti-calnexin, anti-GFP, Alexa Fluor 594 or 488 antibodies(Invitrogen), and DAPI. The right panel shows the phenotype of HeLaKyoto cells following transfection with an RNAi molecule targetingendogenous PARP16 (SEQ ID NO: 43) following staining with ananti-tubulin antibody, Alexa Fluor 594 or 488 antibody (Invitrogen), andDAPI.

FIG. 11 is a set of micrographs showing the co-localization of PARP7-GFPand coilin, and the co-localization of PARP16-GFP and calnexin. In eachexperiment, HeLa Kyoto cells transfected with pEGFP-C1 vectorsexpressing PARP7-GFP or PARP16-GFP were stained with anti-GFP andanti-coilin or anti-calnexin antibodies, and fluorescently labeledsecondary antibodies (Alexa Fluor 594 or 488 antibodies). The figurealso lists a number of protein markers of specific cellular organellesand structures.

FIG. 12 is a diagram of the pcDNA3.1 vector (Invitrogen) showing the CMVpromoter and the restriction sites that may be used for cloning.

FIG. 13 is a diagram of an example of an activity assay using one ormore of the PARP-GFP fusion proteins of the invention.

FIG. 14 is a diagram of an example of an assay for identifying anactivator of one or more PARP-GFP fusion proteins of the invention.

FIG. 15 is a picture of the Bio-Gel P-6 structure and a picture of aCoomassie Blue-stained SDS-PAGE gel showing the use of Bio-Gel P-6 forthe purification of proteins from a crude HeLa Kyoto cell extract. TheSDS-PAGE gel shows the proteins present in cell extract (Extract), incell extract following lectin clarification (Lectin Clarification), inthe lysate prior to passing over the Bio-Gel P-6 resin (Input), in thepellet following centrifugation of the resin (Pellet), and in the eluatefollowing treatment with poly-ADP-ribose glycohydrolase ARH3 (ARH3Release).

FIG. 16 is a set of micrographs showing the co-localization of poly-ADPribose polymers (pADPR) and eIF3, a part of the translation initiationcomplex and a marker of stress granules, in HeLa Kyoto cells followingtreatment with 0 or 250 μM sodium arsenite for 30 minutes andimmunostaining with primary antibodies specific for poly-ADP-ribosepolymers and eIF3, and Alexa Fluor 594 or 488 secondary antibodies(Invitrogen).

FIG. 17 is a set of micrographs showing the co-localization of PARP-GFPfusion proteins with eIF3 in transfected HeLa Kyoto cells followingtreatment with 0 or 250 μM sodium arsenite for 30 minutes. In theseexperiments, HeLa Kyoto cells were transfected with a pEGFP-C1 plasmidexpressing PARP5A-GFP, PARP12-GFP, PARP13.1-GFP, PARP13.2-GFP, orPARP15-GFP fusion protein and treated with 0 or 250 μM sodium arsenite.The cells were fixed and stained with anti-GFP and anti-eIF3, andsecondary fluorescently-labeled antibodies (Alexa Fluor 594 or 488antibodies (Invitrogen)) prior to imaging.

FIG. 18 is a set of micrographs showing the co-localization ofendogenous PARP5A, PARP12, PARP13/13.1, PARP15, or poly-ADP-riboseglycohydrolase (PARG), and eIF3 (a stress granule marker) in HeLa Kyotocells following treatment with 250 μM sodium arsenite for 30 minutes. Inthese experiments, cells were stained with rabbit antibodies specificfor one of PARP5A, PARP12, PARP13/13.1, PARP15, or PARG, and ananti-eIF3 antibody, and fluorescently-labeled secondary antibodies(Alexa Fluor 594 or 488 antibodies (Invitrogen)).

FIG. 19 is a set of micrographs showing the localization ofpoly-ADP-ribose (PAR), endogenous PARP5A, PARP12, PARP13, and PARP15,and eIF3 (a stress granule marker) in hTERT RPE cells followingtreatment with 250 μM sodium arsenite for 30 minutes. In theseexperiments, cells were stained with antibodies specific for one of PAR,PARP5A, PARP12, PARP13, or PARP15, or an anti-eIF3 antibody, and asecondary fluorescently-labeled antibodies (Alexa Fluor 594 or 488antibodies (Invitrogen)).

FIG. 20 is a set of micrographs showing the effect of PARP13.1-GFP,PARP13.2-GFP, or PARP15-GFP overexpression on stress granule formation.In these experiments, HeLa Kyoto cells were transfected with a plasmidexpressing PARP13.1-GFP, PARP13.2-GFP, or PARP15-GFP. The cells werefixed and stained using rabbit anti-GFP and anti-eIF3 antibodies, andfluorescently-labeled secondary antibodies (Alexa Fluor 594 or 488antibodies (Invitrogen)). The cells were also co-stained with DAPI.

FIG. 21 is a set of micrographs showing the co-localization ofPARP11-GFP and eIF3 (a stress granule marker) in HeLa Kyoto cellstransfected with a pEGFP-C1 vector expressing PARP11-GFP followingtreatment with 250 μM sodium arsenite for 30 minutes. Following arsenitetreatment, the cells were immediately fixed and stained using rabbitanti-GFP and anti-eIF3 antibodies, and fluorescently-labeled secondaryantibodies (Alexa Fluor 594 or 488 antibodies (Invitrogen)). The cellswere also stained with DAPI.

FIG. 22 is a set of micrographs showing the effect of PARG99-GFP,PARG102-GFP, or PARG110-GFP overexpression on stress granule formationin HeLa Kyoto cells transfected with a pEGFP-C1 plasmid containing anucleic acid sequence encoding each PARG-GFP fusion protein, followingtreatment with 100 μM sodium arsenite for 30 minutes. Following arsenitetreatment, the cells were fixed and stained with rabbit anti-GFP andanti-eIF3 antibodies, and fluorescently-labeled secondary antibodies(Alexa Fluor 594 or 488 antibodies (Invitrogen)). The images shown inthe right panels show the same data using a threshold filter.

FIG. 23 is a set of micrographs showing the effect of PARG or ARH3knockdown on stress granule formation in HeLa Kyoto cells transfectedwith 30 nM PARG siRNA (CCAGUUGGAUGGACACUAAUU (SEQ ID NO: 34) andUUACGAAGGUACC AUAGAAUU (SEQ ID NO: 35)), ARH3 siRNA(GGACAGAAGCCUUGUACUAUU (SEQ ID NO: 36) and CCAUUGCUGGUGCCUACUAUU (SEQ IDNO: 37)), or a control siRNA (All Stars Negative Control siRNA; QiagenCatalog No. 1027280) following treatment with 100 μM sodium arsenite for30 minutes, or 30 minutes or 1 hour following sodium arsenite washout.The cells were fixed and stained with an anti-eIF3 antibody andsecondary fluorescently-labeled antibodies (Alexa Fluor 594 or 488antibodies) to visualize stress granule formation. The panel on the leftshows an immunoblot of cell lysate from HeLa Kyoto cells treated with 30nM PARG siRNA, ARH3 siRNA, or control siRNA for 48 hours. The immunoblotwas developed using an anti-PARG antibody.

FIG. 24 is a graph showing the percentage of HeLa Kyoto cellstransfected with 30 nM PARG siRNA (SEQ ID NOS: 34 and 35), ARH3 siRNA(SEQ ID NOS: 36 and 37), or a control siRNA (All Stars Negative ControlsiRNA; Qiagen Catalog No. 1027280) containing stress granules followingtreatment with 100 μM sodium arsenite for 30 minutes, or 30 minutes or 1hour following sodium arsenite washout. The cells were fixed and stainedwith a fluorescently-labeled anti-eIF3 antibody to visualize stressgranule formation.

FIG. 25A is a Silver-stained 4-12% SDS-PAGE gel showing the proteinsimmunoprecipitated with an anti-GFP antibody from lysate from HeLa S3cells transfected with a pEGFP-C1 plasmid expressing GFP alone,PARP5A-GFP, PARP12-GFP, PARP13-GFP, PARP13.1-GFP, or PARP15-GFPfollowing treatment with 0 or 250 μM sodium arsenite for 30 minutes.

FIG. 25B is picture of an immunoblot of a 4-12% SDS-PAGE gel containingproteins immunoprecipitated with an anti-GFP antibody from lysate fromHeLa S3 cells transfected with a pEGFP-C1 plasmid expressing GFP,PARP5A-GFP, PARP12-GFP, PARP13-GFP, PARP13.1-GFP, or PARP15-GFPfollowing treatment with 0 or 250 μM sodium arsenite for 30 minutes. Theimmunoblot was developed using a polyclonal anti-poly-ADP-riboseantibody.

FIG. 25C is a picture of several immunoblots of a 4-12% SDS-PAGE gelcontaining proteins immunoprecipitated with an anti-GFP antibody fromlysate from HeLa S3 cells transfected with a pEGFP-C1 plasmid expressingGFP, PARP5A-GFP, PARP12-GFP, PARP13-GFP, PARP13.1-GFP, or PARP15-GFPfollowing treatment with 0 or 20 nM pateamine A for 30 minutes. Theimmunoblots were developed using one of the following antibodies:anti-Ago2, anti-DDX6, anti-LSM1, anti-PABP, anti-FMRP, anti-eIF1A,anti-eIF2α, anti-eIF3η, anti-eIF4A1, and anti-eIF4E.

FIG. 26A is picture of an immunoblot of a 4-12% SDS-PAGE gel containingproteins immunoprecipitated with an anti-GFP antibody from lysate fromHeLa S3 cells transfected with a pEGFP-C1 plasmid expressing TIA1-GFP,PABP-GFP, G3BP-GFP, or Ago2-GFP following treatment with 0 or 20 nMpateamine A for 30 minutes. The immunoblot was developed using apolyclonal anti-poly-ADP-ribose antibody.

FIG. 26B is a picture of an immunoblot of an SDS-PAGE gel containingproteins immunoprecipitated from lysate from untransfected HeLa S3 cellsusing anti-G3BP and anti-Ago2 antibodies following treatment with 0 or250 μM sodium arsenite for 60 minutes. The immunoblot was developedusing a polyclonal anti-poly-ADP-ribose antibody.

FIG. 26C is a picture of an immunoblot of an SDS-PAGE gel containingproteins immunoprecipitated from lysate from HeLa S3 cells transfectedwith a pEGFP-C1 plasmid expressing G3BP1-GFP (full-length), G3BP1-A-GFP(domain A), G3BP1-ABC-GFP (domains A, B, and C), G3BP1-BC-GFP (domains Band C), G3BP1-BCD-GFP (domains B, C, and D), and G3BP1-D-GFP (domain D)following treatment with 0 or 250 μM sodium arsenite for 60 minutes. Theimmunoblot was developed using a polyclonal anti-poly-ADP-riboseantibody.

FIG. 26D is a picture of an immunoblot of an SDS-PAGE gel containingproteins immunoprecipitated from lysate from HeLa S3 cells transfectedwith a pEGFP-C1 plasmid expressing TIA1-GFP (full-length) or TIA1ΔRRM(mutant lacking RRM domain) following treatment with 0 or 250 μM sodiumarsenite for 60 minutes. The immunoblot was developed using a polyclonalanti-poly-ADP-ribose antibody.

FIG. 27 (left panel) is a set of micrographs showing the localization ofpoly-ADP-ribose, and endogenous Ago2 and eIF3 in HeLa cells followingtreatment with 250 μM sodium arsenite for 30 minutes. The cells wereimaged using fluorescently labeled anti-poly-ADP-ribose, anti-Ago2, andanti-eIF3 antibodies. FIG. 26 (right panel) is an immunoblot of a 4-12%SDS-PAGE gel containing proteins immunoprecipitated using an anti-Ago2antibody from untransfected HeLa cells following treatment with 0 or 250μM sodium arsenite for 60 minutes. The immunoblot was developed usinganti-poly-ADP-ribose antibodies.

FIG. 28 is a picture of an immunoblot of a 4-12% SDS-PAGE gel containingproteins immunoprecipitated with an anti-Ago2 antibody from lysate fromuntransfected HeLa cells following treatment with 0 or 250 μM sodiumarsenite for 30 minutes. The immunoblot was developed using a polyclonalanti-PARP13/13.1 antibody.

FIG. 29 is a picture of a Coomassie Blue-stained 4-12% SDS-PAGE gelcontaining proteins immunoprecipitated using an anti-GFP antibody fromlysate from HeLa S3 cells transfected with a pEGFP-C1 plasmid expressingPARP13-GFP following treatment with 0 or 250 μM sodium arsenite for 30minutes.

FIG. 30 is a graph showing the relative expression of luciferase inlysates from 293T cells transfected with a modified pGL4.72[hRlucCP]™vector (Promega); 10 nM of vector-target RNAi (SEQ ID NOS: 38 and 39);and a pEGFP-C1 vector encoding EGFP, G3BP, PAPR5A, PARP12, PARP13,PARP13.1, or PARP15. Luciferase expression was measured in cell lysatesat 48 hours post-transfection. The level of luciferase in treated cellsis compared to the level of luciferase produced in cells transfectedwith the modified pGL4.72[hRlucCP]™ vector alone. As another positivecontrol, the level of luciferase produced from cells transfected withthe modified pGL4.72[hRlucCP]™ vector and the vector-target RNAi isshown (CXCR4 sponge).

FIG. 31 is a graph showing the relative expression of luciferase in 293Tcells transfected with a modified pGL4.72[hRlucCP]™ vector and 20 nM ofnegative RNAi control for PARP13 siRNA (siNeg; All Stars NegativeControl siRNA; Qiagen Catalog No. 1027280) or PARP13 siRNA (siPARP13;GCUCACGGAACUAUGAGCUGAGUUU; SEQ ID NO: 40) following treatment with 0 or30 nM pateamine A for 30 minutes. Luciferase expression was measured incell lysates at 48 hours post-transfection.

DETAILED DESCRIPTION

We have discovered that specific PARP proteins and subsets of PARPproteins have an effect on stress granule formation, nucleation, ordisassembly, and/or are localized in the nucleus and are required forcell cycle progression through mitosis. We have also discovered thatPARGs have an effect on stress granule formation, nucleation, anddisassembly (i.e., stress granule kinetics). The invention thereforeprovides methods, compositions, and kits for the treatment of stressgranule-related disorders and cancer, and methods for determining thepropensity of a subject to develop a stress granule-related disorder orcancer based on these unique activities of specific PARP proteins andPARGs. Conversely, the invention also provides methods for increasingthe proliferation rate of a cell or cell population and increasingstress granule formation in a cell or cell population by modulating PARPfunction, PARG function, or poly-ADP-ribose pathways or homeostasis in acell or cell population. Lastly, the invention provides screeningmethods for the identification of candidate agents that may be usefulfor treating or reducing the likelihood of developing a stressgranule-related disorder and/or cancer.

Methods for Treating a Stress-Granule Related Disorder and DecreasingStress Granule Formation

The present invention provides methods for treating or reducing thelikelihood of developing one or more stress-granule related disorders ina subject. Stress granule-related disorders share a common etiology andpathology linked to oxidative stress and inflammation. Stressgranule-related disorders include, without limitation, cardiovasculardisorders, inflammatory disorders, neurological disorders, orischemic-reperfusion injury.

In the treatment methods provided by the invention, a subject isadministered a therapeutically effective dose of one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP inhibitor(s), one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) PARG activator(s), and/or one or more(e.g., 1, 2, 3, 4, or 5) PARP11 activator(s). A patient receiving thetreatment may be previously diagnosed as having a stress granule-relateddisorder, or may be diagnosed as having a high probability (i.e., atsignificant risk) of developing a stress granule-related disorder. Aperson receiving the treatment may be asymptomatic or may beexperiencing one or more (e.g., 1, 2, 3, 4, or 5) of the symptoms of astress granule-related disorder.

Non-limiting examples of stress granule related disorders include ananeurysm, angina, atherosclerosis, stroke, cerebrovascular disease,congestive heart failure, coronary artery disease, myocardial disease,peripheral vascular disease, granulomatous myocarditis, chronicmyocarditis, myocardial infarction, primary hypertrophic cardiomyopathy,autoimmune diseases, asthma, allergic intraocular inflammatory diseases,arthritis, atopic dermatitis, atopic eczema, cirrhosis, Crohn's disease,ulcerative colitis, diabetes, hemolytic anemia, inflammatory dermatosis,an inflammatory bowel disorder, systemic lupus erythamatosus, psoriasis,rheumatoid arthritis, Wegener's granulomatosis, Hashimoto's thyroiditis,chronic pancreatitis, reactive lymphoid hyperplasia, multiple sclerosis,Alzheimer's disease, Parkinson's disease, Huntingon's disease,amyotrophic lateral sclerosis, retinosa pigmentosum, maculardegeneration, traumatic brain injury, stroke, and peripheral neuropathy.Methods for the diagnosis and monitoring of several stress granulerelated disorders and known in the art. The methods of the invention mayreduce (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100%) one or more (e.g., 1, 2, 3, 4, or 5) of the symptoms of astress granule related-disorder or prevent the onset of one or more(e.g., 1, 2, 3, 4, or 5) of the symptoms of a stress-related disorder.Symptoms of a stress granule-related disorder include, but are notlimited to, pain, swelling, inflammation, loss of cognition, loss ofvision, loss of coordination, difficulty breathing, airway constriction,artery occlusion, diarrhea, elevated blood glucose, increased levels ofpro-inflammatory cytokines, increase protein aggregates or deposits, andincreased cell death (e.g., apoptosis or necrosis). The methods oftreatment provided herein may be used with one or more (e.g., 1, 2, 3,4, or 5) other therapies or therapeutic agents used to treat a stressgranule-related disorder. The effectiveness of treatment may be measuredby a physician using methods known in the art.

In the treatment methods provided herein, a subject may be administeredone or more (e.g., 1, 2, 3, 4, or 5) PARP inhibitors. The one or morePARP inhibitors preferably decrease (e.g., at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or even 100% decrease) the expression (e.g.,protein and/or mRNA levels) and/or one or more activities of PAR5A,PARP12, PARP13.1, PARP13.2, and PARIS. The decrease in expression ofPARP5A, PARP12, PARP13.1, PARP13.2, and PARP15 preferably is a decreasein the expression of one or more nucleic acids containing a sequencehaving at least 80% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%,98%, 99%, or even 100% identity) to PARP5A (SEQ ID NO: 8 or 9), PARP5B(SEQ ID NO: 10), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO: 20), orPARP15 (SEQ ID NO: 22 or 23), or one or more polypeptides encoded bythese nucleic acids. The PARP inhibitors also preferably inhibit one ormore of the activities of a PARP protein (e.g., PARP5A, PARP12,PARP13.1, PARP13.2, or PARP15) including poly-ADP-ribosylation of atarget protein (e.g., a protein in a stress granule, a polypeptideinvolved in the formation or disassembly of a stress granule, or a PARPprotein) or the formation or nucleation of a stress granule. Methods formeasuring the activity of one or more PARP proteins are describedherein.

A subject may also be administered one or more (e.g., 1, 2, 3, 4, or 5)PARG activators. Preferred PARG activators selectively increase theexpression and/or biological activity of PARG protein or ARH3. Forexample, PARG activators desirably increase the level of one or morenucleic acid(s) containing a nucleic acid sequence having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100% identity) to PARG (SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41), orincrease the level of one or more polypeptides encoded by these nucleicacids. Desirably, a PARG activator increases the one or more activitiesof a PARG protein or ARH3, including, but not limited to, hydrolysis ofpoly-ADP-ribose (e.g., poly-ADP-ribose attached to a substrate protein,e.g., a protein localized in a stress granule, a polypeptide involved inthe formation or disassembly of a stress granule, or a PARP protein),the prevention of the assembly of a stress granule, or disassembly of astress granule.

A subject may also be administered one or more (e.g., 1, 2, 3, 4, or 5)PARP11 activators. Desirably, the one or more PARP11 activatorsselectively increase the level of one or more nucleic acids containing asequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100% identity) to PARP11 (SEQ ID NO:17), or increase the level of one or more polypeptides encoded by thesenucleic acids.

Additional desirable PARP11 activators increase one or more activitiesof PARP11, including, but not limited to, poly-ADP-ribosylation of atarget protein (e.g., a protein localized in a stress granule or apolypeptide involved in the formation, a polypeptide involved in thedisassembly of a stress granule, and/or a PARP protein), the preventionof the assembly of a stress granule, or the disassembly of a stressgranule.

Examples of PARP inhibitors that may be used in these methods include anantibody or antibody fragment that selectively binds one or more ofPARP5A, PARP12, PARP13.1, PARP13.2, and PARP15; an RNA aptamer (e.g.,SEQ ID NOS: 40, 49, 99-113, and 122-129); or a small molecule. Examplesof PARG activators that may be used in these methods include one or morenucleic acids containing a sequence having at least 80% sequenceidentity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%identity) to PARG (SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41). Examples ofPARP11 activators that may be used in these methods include one or morenucleic acids containing a nucleic acid sequence having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100% identity) to PARP11 (SEQ ID NO: 17).

In these methods, the one or more PARP inhibitors, one or more PARGactivators, and one or more PARP11 activators may be administeredco-extensively (overlapping bioactive periods) or non-extensively(non-overlapping bioactive periods). In one example, the one or morePARP inhibitors, one or more PARG activators, and one or more PARP11activators may be administered together in a single dose or may beadministered separately in one or more separate doses (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 doses). The amount of one or more PARP inhibitors,one or more PARG activators, and one or more PARP11 activators that maybe administered to a subject per dose may be between 0.1 mg and 1 g, 0.1mg and 750 mg, 0.1 mg and 600 mg, 0.1 mg and 500 mg, 10 mg and 450 mg,10 mg and 400 mg, 10 mg and 350 mg, 10 mg and 350 mg, and 10 mg and 250mg. Various combinations of the one or more PARP inhibitors, one or morePARG activators, and one or more PARP11 activators are contemplatedherein, for example, administration of one or more PARP inhibitorsalone, administration of one or more PARG activators alone,administration of one or more PARP11 activators alone, administration ofone or more PARP inhibitors and one or more PARG activators together,and administration of one or more PARP inhibitors and one or more PARP11activators together.

The one or more PARP inhibitors, one or more PARG activators, and theone or more PARP11 activators may be administered to the subjects once aday, twice a day, three times a day, once a week, twice a week, threetimes a week, four times a week, five times a week, six times a week,seven times a week, bi-weekly, tri-weekly, or monthly. The one or morePARP inhibitors, one or more PARG activators, and the one or more PARP11activators may be administered via the same route of administration orvia different routes of administration. For example, the one or morePARP inhibitors may be administered orally and the PARG activators maybe administered parenterally (e.g., subcutaneously). The one or morePARP inhibitors, one or more PARG activators, and/or the one or morePARP11 activators may be formulated for any known route ofadministration, including oral, intravenous, intraarterial, intraocular,intranasal, intramuscular, and subcutaneous administration. The one ormore PARP inhibitors, one or more PARG activators, and/or the one ormore PARP11 activators may also be formulated for administration in asustained-release form. The therapeutically effective dose of the one ormore PARP inhibitors, one or more PARG activators, and/or one or morePARP11 activators may be determined by a skilled physician using methodsknown in the art, in addition to the in vitro assays described herein.

The invention similarly provides methods of decreasing the number ofstress granules in a cell or a cell population by contacting the cell orcell population with an effective amount of one or more PARP inhibitors,one or more PARG activators, and one or more PARP11 activators (asdescribed for the treatment of subjects above). In these methods, theone or more PARP inhibitors, one or more PARG activators, and the one ormore PARP11 activators are added to the tissue culture medium todecrease the formation of stress granules in a cell or a cellpopulation. Desirably, the cells are cultured for regenerative celltechnology or the cells are primary or germ cells. This method desirablyconfers on the cells protection against oxidative stress and promotesthe longevity and morphology of the cultured cells. Preferred cells thatmay be used in these methods include, without limitation, an epithelialcell, a fibroblast, a kidney cell, a muscle cell, a neuron, ahepatocyte, a sperm, a lymphocyte, or a macrophage. The concentration ofthe one or more PARP inhibitors, the one or more PARG activators, andthe one or more PARP11 activators to be added to the culture medium maybe determined by using the methods described herein (e.g., methods forthe measurement of stress granule formation).

Methods for Treating a Cancer and Decreasing Cell Proliferation

The present invention provides methods for treating or reducing thelikelihood of developing cancer in a subject. All forms of cancer sharea common etiology and pathology of uncontrolled, unregulated, ormisregulated cell proliferation or cell division. Cancers that may betreated by the methods, compositions, and kits of the invention include,without limitation, colon adenocarcinoma, esophagas adenocarcinoma,liver hepatocellular carcinoma, squamous cell carcinoma, pancreasadenocarcinoma, islet cell tumor, rectum adenocarcinoma,gastrointestinal stromal tumor, stomach adenocarcinoma, adrenal corticalcarcinoma, follicular carcinoma, papillary carcinoma, breast cancer,ductal carcinoma, lobular carcinoma, intraductal carcinoma, mucinouscarcinoma, phyllodes tumor, Ewing's sarcoma, ovarian adenocarcinoma,endometrium adenocarcinoma, granulose cell tumor, mucinouscystadenocarcinoma, cervix adenocarcinoma, vulva squamous cellcarcinoma, basal cell carcinoma, prostate adenocarcinoma, giant celltumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma,kidney carcinoma, urinary bladder carcinoma, Wilm's tumor, lymphoma, andnon-Hodgkin's lymphoma. The methods of the invention may reduce (e.g.,at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%) oneor more (e.g., 1, 2, 3, 4, or 5) of the symptoms of a cancer or preventthe onset of one or more (e.g., 1, 2, 3, 4, or 5) of the symptoms of acancer. Symptoms of a cancer include, but are not limited to, blood inurine, pain or burning upon urination, cloudy urine, pain in bone,fractures in bones, fatigue, weight loss, repeated infections, nausea,vomiting, constipation, numbness in the legs, bruising, dizziness,drowsiness, abnormal eye movements, changes in vision, changes inspeech, headaches, thickening of a tissue, rectal bleeding, abdominalcramps, loss of appetite, fever, enlarged lymphnodes, persistent cough,blood in sputum, lung congestion, itchy skin, lumps in skin, abdominalswelling, vaginal bleeding, jaundice, heartburn, indigestion, cellproliferation, and loss of regulation of controlled cell death. Themethods of treatment provided herein may be used with one or more (e.g.,1, 2, 3, 4, or 5) other therapies or therapeutic agents used to treat acancer (e.g., chemotherapy, radiation, and/or surgery). Theeffectiveness of treatment may be measured by a physician using methodsknown in the art. Methods for the diagnosis and monitoring of severalcancers are known in the art.

In the treatment methods provided by the invention, a subject isadministered a therapeutically effective dose of one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) of a PARP inhibitor(s). A patientreceiving the treatment may be previously diagnosed as having a cancer,or may be diagnosed as having a high probability (i.e., at significantrisk) of developing a cancer. A person receiving the treatment may beasymptomatic or may be experiencing one or more of the symptoms of acancer.

In the treatment methods provided herein, a subject may be administeredone or more PARP inhibitors. The one or more PARP inhibitors preferablydecrease (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100% decrease) the expression and/or one or more (e.g., 1, 2, 3, 4,or 5) activities of one or more of PARP1, PARP2, PARP5A, PARP 5B, PARP7,PARP8, PARP14, or PARP16. Preferably, the decrease in expression is adecrease in the level of one or more nucleic acids containing a sequencehaving at least 80% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%,98%, 99%, or even 100% identity) to PARP1 (SEQ ID NO: 1 or 2), PARP2(SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10),PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP14 (SEQ ID NO: 21), orPARP16 (SEQ ID NO: 24), or one or more polypeptides encoded by thesenucleic acids. The PARP inhibitors also preferably inhibit one or moreof the activities of a PARP protein (e.g., PARP1, PARP2, PARP5A, PARP5B,PARP7, PARP8, PARP14, or PARP16) including poly-ADP-ribosylation of atarget protein (e.g., a protein localized in the nucleus or mitoticspindle during cytokinesis, a protein required for progression throughmitosis, and/or a PARP protein) or is required for progression throughmitosis. Methods for measuring the activity of one or more PARP proteinsare described herein.

Examples of PARP inhibitors that may be used in these methods include anantibody or antibody fragment that selectively binds one or more ofPARP1, PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16; an RNAaptamer (e.g., SEQ ID NOS: 43-46, 49, 50, 59-74, 114-121, and 130-136;shown in table below); or a small molecule. In these methods, the one ormore PARP inhibitors may be administered co-extensively (overlappingbioactive periods) or non-extensively (non-overlapping bioactiveperiods). In one example, the one or more PARP inhibitors may beadministered together in a single dose or may be administered separatelyin one or more separate doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10doses). The amount of one or more PARP inhibitors may be between 0.1 mgand 1 g, 0.1 mg and 750 mg, 0.1 mg and 600 mg, 0.1 mg and 500 mg, 10 mgand 450 mg, 10 mg and 400 mg, 10 mg and 350 mg, 10 mg and 350 mg, and 10mg and 250 mg. Various combinations of the one or more PARP inhibitorsare contemplated herein, for example, administration of one or more PARPinhibitors alone or co-administration of one or more PARP inhibitorswith another therapeutic for the treatment of cancer or a therapeuticused to alleviate one or more symptoms of a cancer.

TABLE 1 Specific PARP RNAi molecules PARP1 AAGCCUCCGCUCCUGAACAAUSEQ ID NO: 44 PARP2A AAUCAGUGUAAUGAACUACUA SEQ ID NO: 45 PARP2BAAUGAUUCAGCUAUUAGAAGA SEQ ID NO: 46 PARP3 GGACCCAGGUGUAUGAGGACUACAASEQ ID NO: 47 PARP4 AAACAAGGAUUUCUACUAAGA SEQ ID NO: 48 PARP5AAACAAUUCACCGUCGUCCUCU SEQ ID NO: 49 PARP5B AAGCUUCAGAAUGGUGCAAAUSEQ ID NO: 50 PARP6 CCCAACAAUGGAAACAUCUGAGCAA SEQ ID NO: 51 PARP6UUGCUCAGAUGUUUCCAUUGUUGGG SEQ ID NO: 52 PARP6 GGUUCAAGGCAAGUGGUACCAUCAASEQ ID NO: 53 PARP6 UUGAUGGUACCACUUGCCUUGAACC SEQ ID NO: 54 PARP6CAAAGUGGAAGUGUUUGGCUACCCU SEQ ID NO: 55 PARP6 AGGGUAGCCAAACACUUCCACUUUGSEQ ID NO: 56 PARP6 CAGAACAGAGGAUUCCAACAUUGAA SEQ ID NO: 57 PARP6UUCAAUGUUGGAAUCCUCUGUUCUG SEQ ID NO: 58 PARP7 UGAGGUCUUUGAGGCCAAUAUUAAASEQ ID NO: 59 PARP7 UUUAAUAUUGGCCUCAAAGACCUCA SEQ ID NO: 60 PARP7GACUUUCUGCAAGGCACUUGUAUUU SEQ ID NO: 61 PARP7 AAAUACAAGUGCCUUGCAGAAAGUCSEQ ID NO: 62 PARP7 UCCUCCACCUCUUGAAGCAACUUCA SEQ ID NO: 63 PARP7UGAAGUUGCUUCAAGAGGUGGAGGA SEQ ID NO: 64 PARP7 AAUGAUGACCAGAGUUACCCUUAUUSEQ ID NO: 65 PARP7 AAUAAGGGUAACUCUGGUCAUCAUU SEQ ID NO: 66 PARP8GGAAGAUUCUGAAGGUGACAAUGAU SEQ ID NO: 67 PARP8 AUCAUUGUCACCUUCAGAAUCUUCCSEQ ID NO: 68 PARP8 CCCACAACUGGAAGCUGAUUUGUCA SEQ ID NO: 69 PARP8UGACAAAUCAGCUUCCAGUUGUGGG SEQ ID NO: 70 PARP8 GAAGUGGAAUCUAUCUUAGUCCAAUSEQ ID NO: 71 PARP8 AUUGGACUAAGAUAGAUUCCACUUC SEQ ID NO: 72 PARP8GCCUUAUGUGAAGUGAUCACCUCAU SEQ ID NO: 73 PARP8 AUGAGGUGAUCACUUCACAUAAGGCSEQ ID NO: 74 PARP9 GCCGGAGCAGCAGCUUACAAUGAAA SEQ ID NO: 75 PARP9UUUCAUUGUAAGCUGCUGCUCCGGC SEQ ID NO: 76 PARP9 CCCUCUGAAUUUGUGUACAAAGACUSEQ ID NO: 77 PARP9 AGUCUUUGUACACAAAUUCAGAGGG SEQ ID NO: 78 PARP9GGACCCUACUGUUGCUGCCUUUAAA SEQ ID NO: 79 PARP9 UUUAAAGGCAGCAACAGUAGGGUCCSEQ ID NO: 80 PARP9 UGGCAGACGGCAGAUGUAAUUGUUA SEQ ID NO: 81 PARP9UAACAAUUACAUCUGCCGUCUGCCA SEQ ID NO: 82 PARP10 CAUGGUGCAGGGUAGAGGGAUUAUGSEQ ID NO: 83 PARP10 CAUAAUCCCUCUACCCUGCACCAUG SEQ ID NO: 84 PARP10GCCUGGUGGAGAUGGUGCUAUUGAU SEQ ID NO: 85 PARP10 AUCAAUAGCACCAUCUCCACCAGGCSEQ ID NO: 86 PARP10 AGACGUCGCUCUCUUGCCACUUGAA SEQ ID NO: 87 PARP10UUCAAGUGGCAAGAGAGCGACGUCU SEQ ID NO: 88 PARP10 UGGGCAGCAUUAGCUGCCAUGUGUUSEQ ID NO: 89 PARP10 AACACAUGGCAGCUAAUGCUGCCCA SEQ ID NO: 90 PARP11CAACAAACAAUGAAGUGGAUGACAU SEQ ID NO: 91 PARP11 AUGUCAUCCACUUCAUUGUUUGUUGSEQ ID NO: 92 PARP11 CAGCCGGAUACCAACAGUCAGUGUU SEQ ID NO: 93 PARP11AACACUGACUGUUGGUAUCCGGCUG SEQ ID NO: 94 PARP11 CAAACCCUUGUGGCUCCAUUUCUUUSEQ ID NO: 95 PARP11 AAAGAAAUGGAGCCACAAGGGUUUG SEQ ID NO: 96 PARP11UGCCACCACACUGGGAGAAUGUGAA SEQ ID NO: 97 PARP11 UUCACAUUCUCCCAGUGUGGUGGCASEQ ID NO: 98 PARP12 UCCACCUCUGCAGGUUCAUGGUCUA SEQ ID NO: 99 PARP12UAGACCAUGAACCUGCAGAGGUGGA SEQ ID NO: 100 PARP12UGCCAGAAAUUUGCCAACAUUACAA SEQ ID NO: 101 PARP12UUGUAAUGUUGGCAAAUUUCUGGCA SEQ ID NO: 102 PARP12GGUGAGCAGGCUGCCUACCAUUUAU SEQ ID NO: 103 PARP12AUAAAUGGUAGGCAGCCUGCUCACC SEQ ID NO: 104 PARP12AGGAUUUGGACAACAUGGAACUUAU SEQ ID NO: 105 PARP12AUAAGUUCCAUGUUGUCCAAAUCCU SEQ ID NO: 106 PARP13GCUGACCCAAGAGUAGCACUUGUUA SEQ ID NO: 107 PARP13UAACAAGUGCUACUCUUGGGUCAGC SEQ ID NO: 108 PARP13CCGGUGGCAGAUGCUUAUUGGUAAA SEQ ID NO: 109 PARP13UUUACCAAUAAGCAUCUGCCACCGG SEQ ID NO: 110 PARP13GCUCACGGAACUAUGAGCUGAGUUU SEQ ID NO: 40 PARP13 AAACUCAGCUCAUAGUUCCGUGAGCSEQ ID NO: 111 PARP13 UGCCUCAGUGGUAUGUGCAGCAGAU SEQ ID NO: 112 PARP13AUCUGCUGCACAUACCACUGAGGCA SEQ ID NO: 113 PARP14UGGCCUGUCUAAUGAUGACUUUCAA SEQ ID NO: 114 PARP14UUGAAAGUCAUCAUUAGACAGGCCA SEQ ID NO: 115 PARP14CCUGGUGCUGAUGACUACAGUUUAA SEQ ID NO: 116 PARP14UUAAACUGUAGUCAUCAGCACCAGG SEQ ID NO: 117 PARP14GCCACUUUCUGUGUUCCCAUACUAU SEQ ID NO: 118 PARP14AUAGUAUGGGAACACAGAAAGUGGC SEQ ID NO: 119 PARP14GAAGAGUCACUAGAUCUUCCCUUAU SEQ ID NO: 120 PARP14AUAAGGGAAGAUCUAGUGACUCUUC SEQ ID NO: 121 PARP15GAUGAAUUCACUAACUGGUCAAGAA SEQ ID NO: 122 PARP15UUCUUGACCAGUUAGUGAAUUCAUC SEQ ID NO: 123 PARP15CCUAUCACAGUUGCUGAUAACAUAA SEQ ID NO: 124 PARP15UUAUGUUAUCAGCAACUGUGAUAGG SEQ ID NO: 125 PARP15GGACUGACAUGAAUCAUCAGCUGUU SEQ ID NO: 126 PARP15AACAGCUGAUGAUUCAUGUCAGUCC SEQ ID NO: 127 PARP15CGAGUACUUACUGGAGUCUUCACAA SEQ ID NO: 128 PARP15UUGUGAAGACUCCAGUAAGUACUCG SEQ ID NO: 129 PARP16CAGUGCAGGGAAGGCAGAGUUUGAA SEQ ID NO: 130 PARP16UUCAAACUCUGCCUUCCCUGCACUG SEQ ID NO: 131 PARP16GAGACCAAAGGAGAACGAGACCUAA SEQ ID NO: 132 PARP16UUAGGUCUCGUUCUCCUUUGGUCUC SEQ ID NO: 133 PARP16GACUUGAGCCUGGCCCUCAUAUACA SEQ ID NO: 134 PARP16UGUAUAUGAGGGCCAGGCUCAAGUC SEQ ID NO: 135 PARP16CCCAAGUACUUCGUGGUCACCAAUA SEQ ID NO: 43 PARP16 UAUUGGUGACCACGAAGUACUUGGGSEQ ID NO: 136

The one or more PARP inhibitors may be administered to the subjects oncea day, twice a day, three times a day, once a week, twice a week, threetimes a week, four times a week, five times a week, six times a week,seven times a week, bi-weekly, tri-weekly, or monthly. The one or morePARP inhibitors may be administered via the same route of administrationor via different routes of administration. For example, the one or morePARP inhibitors may be administered orally and one or more other PARPinhibitors may be administered parenterally (e.g., subcutaneously). Theone or more PARP inhibitors may be formulated for any known route ofadministration, including oral, intravenous, intraarterial, intraocular,intranasal, intramuscular, and subcutaneous administration. The one ormore PARP inhibitors may also be formulated for administration in asustained-release form.

The invention similarly provides methods of decreasing the proliferationrate in a cell or a cell population by contacting the cell or cellpopulation with an effective amount of one or more PARP inhibitors (asdescribed for the treatment of subjects above). In these methods, theone or more PARP inhibitors are added to the tissue culture medium todecrease the proliferation rate of a cell or a cell population.Desirably, the cells are cultured for regenerative cell technology, thecells are primary or germ cells, or the cells are being stored for latertherapeutic or experimental use. This method desirably confers on thecells protection from oxidative stress, promotes the longevity andmorphology of the cultured cells, and/or delays the onset of senescenceof the cells. Preferred cells that may be used in these methods include,without limitation, an epithelial cell, a fibroblast, a kidney cell, amuscle cell, a neuron, a hepatocyte, a sperm, a lymphocyte, or amacrophage. The concentration of the one or more PARP inhibitors may bedetermined using methods known in the art and those methods describedherein (e.g., methods for the measurement of cell growth and division).

Pharmaceutical Compositions

The invention further provides pharmaceutical compositions for treatingor reducing the likelihood of developing one or more stress granuledisorders and cancer. For example, the compositions for the treating ordecreasing the likelihood of developing a stress granule-relateddisorder may include one or more of the PARP inhibitors (e.g., PARP5A,PARP12, PARP13.1, PARP13.2, and PARP15 inhibitors), one or more of thePARG activators (e.g., PARG protein and ARH3 activators), and/or one ormore of the PARP11 activators as described above. Compositions fortreating or decreasing the likelihood of developing a cancer may includeone or more of the PARP inhibitors (e.g., PARP1, PARP2, PARP5A, PARP5B,PARP7, PARP8, PARP14, and PARP16 inhibitors) as described above. The oneor more agents that may be used as pharmaceutical compositions fortreating or reducing the likelihood of developing a stressgranule-related disorder or cancer may also be identified using thescreening assays provided herein.

Examples of pharmaceutical compositions for treating or reducing thelikelihood of developing a stress granule related disorder include oneor more of an antibody or antibody fragment that specifically binds toPARP5A, PARP12, PARP13.1, PARP13.2, and PARP15; an RNAi molecule thatdecreases the expression of one or more of PARP5A, PARP12, PARP13.1,PARP13.2, and PARP15 (e.g., SEQ ID NOS: 40, 49, 99-113, and 122-129);one or more nucleic acids containing a sequence having at least 80%sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, oreven 100%) to PARP11 (SEQ ID NO: 17), PARG (SEQ ID NO: 42), or ARH3 (SEQID NO: 41), and one or more small molecules or metabolites identified inthe screening assays provided herein.

Examples of pharmaceutical compositions for treating or reducing thelikelihood of developing a cancer include one or more of an antibody orantibody fragment that specifically binds to PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16; an RNAi molecule thatdecreases the expression of one or more of PARP1, PARP2, PARP5A, PARP5B,PARP7, PARP8, PARP14, and PARP16 (e.g., SEQ ID NOS: 43-46, 49, 50,59-74, 114-121, and 130-136); and one or more small molecules ormetabolites identified in the screening assays provided herein.

The pharmaceutical compositions provided by the invention may furtherinclude one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) secondaryagents. Non-limiting examples of secondary agents that may be includedin the compositions of the invention are one or more chemotherapeuticagent(s), one or more non-steroidal anti-inflammatory drug(s), one ormore immunosuppressive agent(s), one or more calcineurin inhibitor(s),or one or more analgesic(s). Examples of these classes of therapeuticagents are known in the art.

The dose of one or more PARP inhibitors, PARP11 activators, and/or PARGactivators may be between 0.1 mg and 1 g, 0.1 mg and 750 mg, 0.1 mg and600 mg, 0.1 mg and 500 mg, 10 mg and 450 mg, 10 mg and 400 mg, 10 mg and350 mg, 10 mg and 350 mg, and 10 mg and 250 mg.

The dose of one or more secondary agents that may be included in thecompositions of the invention may be between 0.1 mg and 2 g, 0.1 mg and1.5 mg, 0.1 mg and 1 g, 0.1 mg and 750 mg, 1 mg and 650 mg, 1 mg and 550mg, 1 mg and 500 mg, 10 mg and 450 mg, 10 mg and 400 mg, 10 mg and 350mg, 10 mg and 350 mg, and 10 mg and 250 mg.

The compositions may be formulated using any known method includingformulation as a pill, an injectible fluid (e.g., in PBS), or in asustained-release form. The compositions may be formulated for anyspecific route of administration including oral, intramuscular,intraocular, intranasal, subcutaneous, intraarterial, and intravenousadministration.

Kits

The invention further provides kits containing one or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) of the pharmaceutical compositions describedherein. The kits may further contain materials to aid in theadministration of the pharmaceutical agents (e.g., a syringe). The kitsmay contain one or more doses of a pharmaceutical agent provided by theinvention. The kits may further contain instructions for administeringthe pharmaceutical compositions to a subject having a stressgranule-related disorder or cancer, or a subject that has a highprobability of developing (a high propensity) for developing a stressgranule-related disorder or cancer.

Medical Screening Assays

The invention further provides methods of determining the propensity ofa subject to develop a stress granule-related disorder by determiningthe expression (e.g., protein or mRNA levels) and/or one or more (e.g.,1, 2, 3, 4, or 5) activities of one or more (e.g., 1, 2, 3, 4, 5, 6, 7,or 8) of PARP5A, PARP11, PARP12, PARP13.1, PARP13.2, PARP15, PARG, andARH3 in a subject, wherein an increase (e.g., at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) in the expression (e.g.,mRNA and/or protein) and/or one or more (e.g., 1, 2, 3, 4, or 5)activities of one or more (e.g., 1, 2, 3, 4, or 5) of PARP5A, PARP12,PARP13.1, PARP13.2, and PARP15, and/or a decrease (e.g., at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%) in one or more(e.g., 1, 2, or 3) of PARG, ARH3, and PARP11 expression (e.g., proteinor mRNA levels) and/or one or more activities indicates an increasedpropensity to develop a stress-granule related disorder. In this method,the expression measured may be the level of one or more nucleic acidscontaining a nucleic acid sequence having at least 80% (e.g., at least85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to PARP5A (SEQ ID NO: 8or 9), PARP5B (SEQ ID NO: 10), PARP11 (SEQ ID NO: 17), PARP13.1 (SEQ IDNO: 19), PARP13.2 (SEQ ID NO: 20), PARP15 (SEQ ID NO: 22 or 23), PARG(SEQ ID NO: 42), or ARH3 (SEQ ID NO: 41), or one or more polypeptidesencoded by these nucleic acids.

The invention further provides methods of determining the propensity ofa subject to develop a cancer by determining the expression (e.g.,protein and/or mRNA level) and/or one or more activities (e.g., 1, 2, 3,4, or 5) of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of PARP1,PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16 in a subject,wherein an increase (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or even 100%) in the expression and/or one or more activitiesof one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of PARP1, PARP2,PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16 indicates an increasedpropensity to develop cancer. In this method, the expression measuredmay be the level of one or more nucleic acids containing a nucleic acidsequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or even 100%) identity to PARP1 (SEQ ID NO: 1 or 2), PARP2(SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP14 (SEQ ID NO: 21), andPARP16 (SEQ ID NO: 16), or the level of one or more polypeptides encodedby these nucleic acids.

In each of these methods, the level of expression of a nucleic acid maybe determined using RT-PCR and the level of a polypeptide encoded by anucleic acid may be determined by a variety of different antibody-basedtechniques including, but not limited to, immunoblotting,fluorescence-assisted cell sorting (FACS), and enzyme-linkedimmunosorbent assay (ELISA). In each assay, the level of expression in asubject may be compared to a subject known to have a stressgranule-related disorder or cancer, or a control subject that does nothave a stress granule-related disorder or cancer, or a very lowpropensity for developing a stress granule-related disorder or cancer.

PARP, PARG, and ARH3 Fusion Proteins

General Design

The invention provides fusion proteins for each PARP, PARG, and ARH3.The fusion proteins may be used to identify unique biological activitiesfor each PARP, PARG, and ARH3 protein and to identify specificinhibitors and activators for each PARP, PARG, or ARH3, or specificsubsets of these proteins. The invention provides nucleic acid sequencesencoding these PARP, PARG, and ARH3 fusion proteins. The nucleic acidscontain a sequence that is at least 80% identical (e.g., at least 85%,90%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) to the full-lengthsequence of PARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3), PARP3 (SEQID NO: 4), PARP3.2 (SEQ ID NO: 5), PARP3.3 (SEQ ID NO: 6), PARP4 (SEQ IDNO: 7), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP6 (SEQID NO: 11), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP9 (SEQ IDNO: 14), PARP10 (SEQ ID NO: 15), PARP10.2 (SEQ ID NO: 16), PARP11 (SEQID NO: 17), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2(SEQ ID NO: 20), PARP14 (SEQ ID NO: 21), PARP15.1 (SEQ ID NO: 22),PARP15.2 (SEQ ID NO: 23), PARP16 (SEQ ID NO: 24), PARG (SEQ ID NO: 42),or ARH3 (SEQ ID NO: 41).

The nucleic acids of the invention further contain nucleic acidsequences encoding one or two polypeptide tags. The nucleic acidsencoding a polypeptide tag may be placed at a position 5′ or a position3′ to the sequence encoding a PARP, PARG, or ARH3 protein. For example,the 3′ end of a nucleic acid sequence encoding a polypeptide tag maydirectly abut (i.e., no intervening nucleotides) the 5′ end of a nucleicacid sequence encoding a PARP, PARG, or ARH3 protein. In anotherexample, the 5′ end of a nucleic acid sequence encoding a polypeptidetag may directly abut (i.e., no intervening nucleotides) the 3′ end ofnucleic acid sequence encoding a PARP, PARG, or ARH3 protein. In anotherexample, one or more nucleotides (e.g., at least 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, or 400nucleotides) separate the 5′ end of the sequence encoding thepolypeptide tag from the 3′ end of the sequence encoding a PARP protein,or separate the 3′ end of the sequence encoding the polypeptide tag fromthe 5′ end of the sequence encoding the PARP protein. Sequences encodingthe polypeptide tags are described in further detail below.

Polypeptide Tags

Polypeptide tags may be attached to a native protein sequence in orderto aid in the purification of the protein, to label the protein forvisualization in the cell, and/or to increase the thermodynamicstability and/or half-life of a protein. Nucleic acids encoding apolypeptide tag(s) may include one or more of the following sequences: asequence encoding an epitope which may be recognized by a specificantibody recognizing the epitope (e.g., 1, 2, 3, 4 or 5 antigenicpeptide sequences); a sequence encoding a protein that is bound withhigh affinity by a specific binding partner; one or more (e.g., 1, 2, 3,4, or 5) sequence(s) encoding a peptide sequence that aids inpurification (e.g., a His₆ tag); one or more (e.g., 1, 2, 3, 4, 5, 6, or7) sequence(s) encoding a protease recognition sequence; and one or more(e.g., 1, 2, 3, or 4) sequences encoding a protein or a domain of aprotein which increases the thermodynamic stability or half-life of theprotein. The size of the nucleic acid sequence encoding the polypeptidetag may be between 1-50 nucleotides, 1-100 nucleotides, 1-200nucleotides, 1-300 nucleotides, 1-400 nucleotides, 1-500 nucleotides,200-500 nucleotides, 1-1,000 nucleotides, 1-5,000 nucleotides, 1-8,000nucleotides, 1-10,000 nucleotides, or 1-20,000 nucleotides. Severalpolypeptide tags and sequences encoding polypeptide tags are known inthe art. Non-limiting examples of sequences that may be incorporated inpolypeptide tags are described below.

The nucleic acids encoding a polypeptide tag may contain sequences forone or more (e.g., 1, 2, 3, 4, or 5) epitopes or antigenic peptidesequences. Epitopes incorporated into polypeptide tags may be used toaid in the purification of a fusion protein, for e.g., by use of anantibody that specifically binds to the epitope. Examples of epitopesequences include, but are not limited to, a FLAG peptide (DYKDDDDK; SEQID NO: 30); a glutathione-S-transferase (GST) peptide; a KT3 peptide(KPPTPPPEPET; SEQ ID NO: 31); a hemagglutinin peptide (YPYDVPDYA; SEQ IDNO: 32), a calmodulin-binding peptide (Methods in Molecular Biology: E.coli Gene Expression Protocols, volume 205, Humana Press, 2003, pp.79-97), a R-tag peptide (Jones et al., Protein Expr. Purif. 53:404-410,2007), a V5 peptide, a c-myc peptide, and peptides derived fromchitin-binding protein (CBP), CYD, Strep II, HPC, and maltose bindingprotein (MBP), as described in Lichty et al. (Protein Expr. Purif.41:98-105, 2005).

Nucleic acids encoding a polypeptide tag may contain sequences for oneor more (e.g., 1, 2, 3, 4, or 5) proteins with specific bindingpartners. Desirably, the specific binding partner has a high affinity(e.g., K_(D)<150 nM or K_(D)<250 nM) to the peptide sequence in thepolypeptide tag. Non-limiting examples of sequences that encode aprotein with a high-affinity binding partner is biotin and the ZZ-domainof S. aureus protein A (e.g., a nucleic acid sequence with at least 80%identity to SEQ ID NO: 27). Additionally, the polypeptide tag maycontain one or more peptide sequences that aid in the purification ofthe protein. Non-limiting examples of peptide sequences that aid in thepurification of a protein include a His₆ tag, chitin-binding protein(CBP), maltose-binding protein (MBP), and glutathione-S-transferase(GST). For example, a protein containing a polypeptide tag containing aHis₆ tag may be purified by passing a crude cellular lysate over a metalmatrix (e.g., a Ni⁺-Sepharose resin).

A polypeptide tag may also contain a sequence encoding a protein thatincreases the thermodynamic stability, half-life, and/or solubility of aprotein. Non-limiting examples of peptides that increase the solubilityof a protein include thioredoxin and poly(NANP). Additional non-limitingexamples of proteins that increase the thermodynamic stability orhalf-life of a protein include the Fc domain of an antibody and albumin.A polypeptide tag may also contain one or more (e.g., 1, 2, 3, or 4)sequences encoding a protein that allows for the visualization of thefusion protein in the cell (e.g., a polypeptide tag containing asequence encoding a fluorescent protein, such as green fluorescenceprotein).

A polypeptide tag may also contain one or more (e.g., 1, 2, 3, 4, 5, 6,or 7) protease recognition sequences. A fusion protein may be treatedwith one or more (e.g., 1, 2, 3, or 4) specific proteases that cleavethe fusion protein at the one or more specific protease recognitionsequences at any step in the purification process (e.g., after beingbound to a resin or solid surface) to remove the polypeptide tag(s) fromthe remainder of the fusion protein. Non-limiting examples of proteaserecognition sequences include TEV protease (Glu-X-X-Tyr-X-Gln-Ser; SEQID NO: 26), factor Xa (Ile-Glu/Asp-Gly-Arg), Ala-64 subtilisin(Gly-Ala-His-Arg), clostripain (Arg and Lys-Arg), collagenase(Pro-Val-Gly-Pro), enterokinase (Asp-Asp-Asp-Asp-Lys), renin(Pro-Phe-His-Leu-Leu), and α-thrombin (Leu-Val-Pro-Arg-Gly-Ser). When apolypeptide tag is present at the N-terminus of a fusion protein, aprotease recognition sequence is preferably located at a position 3′ toa peptide sequence encoding an epitope, a sequence encoding a proteinthat is bound with high affinity by a specific binding partner, or asequence encoding a peptide sequence that aids in purification. When apolypeptide tag is present at the C-terminus of a fusion protein, aprotease recognition sequence is preferably located at a position 5′ toa peptide sequence encoding an epitope, a sequence encoding a proteinthat is bound with high affinity by a specific binding partner, and/or asequence encoding a peptide sequence that aids in purification. Apolypeptide tag may contain one or more (e.g., 1, 2, 3, 4, 5, 6, or 7)of the same or different protease recognition sequences in tandem (i.e.,without intervening amino acids) or with one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) intervening amino acids between each proteaserecognition sequence. Methods for the treatment of a fusion proteincontaining a protease recognition sequence in the polypeptide tag withone or more protease(s) are known in the art.

Expression Vectors

A number of expression vectors for the expression of a nucleic acidencoding one or more nucleic acids encoding a PARP, PARG, and/or ARH3fusion protein of the invention are known in the art. Different examplesof expression vectors are available for expression of the PARP, PARG,and/or ARH3 fusion proteins in mammalian cells, insect cells, yeastcells, and bacterial cells. For example, the pEGFP-C1 mammalian vector(Invitrogen) contains a CMV promoter sequence, a nucleic acid sequenceencoding green fluorescence protein, a multiple cloning site forinsertion of nucleic acid sequence encoding a PARP, PARG, or ARH3nucleic acid (e.g., a sequence with 80% to one of SEQ ID NOS: 1-24, 41,and 42). Additional non-limiting examples of publicly-availablemammalian expression vectors include constitutive expression vectorsGateway® pDEST™26, pDEST™27, pDEST™40, and pDEST™47 (Invitrogen);adenoviral expression vectors (e.g., pAd/CM/V5-Dest Gateway® Vector Kit(Invitrogen); episomal expression vectors pCEP4 and pEBNA DEST(Invitrogen); lentiviral expression vectors (e.g., ViraPower™ Bsd;Invitrogen); and regulated expression vectors Gateway® pT-Rex™-DEST 30and pT-Rex™-DEST 31 (Invitrogen). Non-limiting examples of bacterialexpression vectors include Gateway® pDEST™14; Gateway® pDEST™15;Gateway® pDEST™17; Gateway® pDEST™24; Gateway® pET-DEST42; pEM7/Bsd;pEM7/Zeo; pRSET A, B, & C; pRSET-BFP; pRSET-CFP; pRSET-EmGFP; pTrcHIs A,B, & C; and pTrcHIs2 A, B, & C vectors (Invitrogen). Non-limitingexamples yeast expression vectors include pAO815; pGAPZ A, B, & C;pPIC3.5K; pPIC9K; pTEF1/Bsd; pTEF1/Zeo; pYC2/CT; pYES2; pYES2/CT; andpYES3/CT (Invitrogen). Non-limiting examples of insect and baculovirusexpression vectors include Gateway® pDEST™10; Gateway® pDEST™20;Gateway® pDEST™8; Gateway® pMT-DEST™48; pAC5.1/V5-His A, B, & C;pFastBac Dual; and pIB/V5-His-DEST (Invitrogen).

The expression vectors used to express a fusion protein may include oneor more (e.g., 1, 2, 3, 4, or 5) constitutive promoter sequences and/orone or more (e.g., 1, 2, 3, 4, or 5) inducible promoter sequences.Non-limiting examples of constitutive promoter sequences includebacterial promoters (e.g., E. coli σ ⁷⁰, σ^(S), σ³², or σ⁵⁴ promoters;B. subtilis σ ^(A) or σ^(B) promoters; T7 RNA polymerase-basedpromoters; and a bacteriophage SP6 promoter), yeast promoters (e.g.,pCyc, pAdh, pSte5, ADH1, cyc100, cyc70, cyc43, cyc28, cyc16, pPGK1,pCYC, GPD (TDH3), and CLB1 promoters), and mammalian promoters (e.g.,cytomegalovirus immediate early gene-based promoters, SV40 earlypromoter, and Rous sarcoma virus promoter). Non-limiting examples ofinducible promoter sequences include alcohol dehydrogenase I genepromoters, tetracycline-responsive promoter systems, glucocorticoidreceptor promoters, estrogen receptor promoter, ecdysone receptorpromoters, metallothionein-based promoters, and T7-polymerase basedpromoters. Several different mammalian expression vectors available thatallow for the inducible expression of a nucleic acid sequence (e.g., aPARP fusion protein) are publicly available including pTet-On-Advanced(Clontech), pERV3 (Stratagene), pNEBR-R1 (New England BioLabs), andpCMV5-CymR (Qbiogene).

PARP, PARG, and ARH3 Proteins

The above-described methods for the generation of PARP, PARG, or ARH3fusion proteins may be modified to generate PARP, PARG, or ARH3proteins. In these methods, the expression vectors that contain anucleic acid sequence encoding a PARP, PARG, or ARH3 fusion protein maybe modified to remove the nucleic acid sequences encoding thepolypeptide tag. The modified vector may then be introduced into a cellto generate a transgenic cell for the expression of the full-length orwild-type PARP, PARG, or ARH3 protein. The produced PARP, PARG, or ARH3protein may contain one or more post-translational modifications,including phosphorylation and poly-ADP-riboyslation. Thepost-translational modifications may be introduced using recombinantenzymes in vitro or may be the result of processing within thetransgenic cell.

Methods for the expression and purification of one or more PARP, PARG,and ARH3 proteins are the same as those employed for the correspondingPARP, PARG, and ARH3 fusion proteins, with the exception that affinitypurification using antibodies and molecules that specifically recognizethe polypeptide tag will not be employed.

Transgenic Cells and Mammals

One or more nucleic acids encoding a PARP, PARG, and/or ARH3 protein,and/or PARP, PARG, and/or ARH3 fusion protein may be introduced into atransgenic cell using methods known in the art, including, but notlimited to electroporation, microinjection, lipid-mediated transfection(e.g., liposomal delivery systems), calcium phosphate-mediatedtransfection, DEAE dextran-mediated transfection, DNA transfection bybiolistics, DNA transfection mediated by polybrene, and virus-mediatedtransduction.

The one or more nucleic acids encoding a PARP, PARG, and/or ARH3protein, and/or PARP, PARG, and/or ARH3 fusion protein may be introducedinto any type of cell, including, but not limited to, a mammalian cell(e.g., a human, mouse, rat, monkey, or rabbit cell), a yeast cell, abacterial cell, or an insect cell. A mammalian cell that expresses oneor more nucleic acids encoding a PARP, PARG, and/or ARH3 protein, and/orPARP, PARG, and/or ARH3 fusion protein may include a fibroblast, anepithelial cell, an endothelial cell, a smooth muscle cell, ahepatocyte, a kidney cell, and a lymphocyte. Additional examples ofsuitable mammalian cell lines include COS-7 monkey kidney cells, CV-1, Lcells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, humanembryonic kidney (HEK) cells, HeLa cells (e.g., HeLa S3 or HeLa Kyotocells), 293 cells, 293T cells, and BHK cell lines. One or more nucleicacids may also be expressed in a cell (e.g., a mammalian cell, abacterial cell, or a yeast cell) that has been engineered to express oneor more (e.g., 1, 2, 3, or 4) chaperone proteins, one or more (e.g., 1,2, 3, or 4) enzymes that promote the post-translational modification ofproteins, and/or contain one or more (e.g., 1, 2, 3, or 4) mutations inthe nucleic acids encoding one or more (e.g., 1, 2, 3, or 4) proteinsthat have a negative effect on the expression of a transgenic protein(e.g., a PARP fusion protein), such as a specific RNAse or protease. Anexample of a bacterial cell that has been engineered to contain amutation in a RNAse is BL21 Star™ (Invitrogen). A variety of cells arecommercially available for the expression of one or more recombinantproteins (e.g., one or more PARP, PARG, and/or ARH3 fusion proteins),including, but not limited to, bacterial competent cells (e.g., BL21-AI™One Shot®, One Shot®-BL21(DE3), and One Shot®-BL21(DE3) pLysE, One Shot®BL21(DE3) pLysS (Invitrogen); and mammalian competent cells (e.g.,Espresso Competent Hela S3 Cells, Espresso Competent CHO-K1 cells, andEspresso Competent HEK 293 cells (Neuromics), MaxPAK Competent HeLa S3cells, MaxPAK Competent CHO-K1 cells, and MaxPAK Competent HEK 293 cells(Genlantis)).

A transgenic cell that contains one or more nucleic acids encoding aPARP, PARG, and/or ARH3 protein, and/or PARP, PARG, and/or ARH3 fusionprotein may a stable cell line (e.g., a cell that has integrated the oneor more nucleic acids encoding a PARP fusion protein into one or more ofits chromosomes). Alternatively, a transgenic cell may contain the oneor more nucleic acids encoding a PARP, PARG, and/or ARH3 protein, and/orPARP, PARG, and/or ARH3 fusion protein in a plasmid or on an artificialchromosome, which replicates independently of the chromosomes of thecell.

A transgenic mammal may also be produced from a transgenic cellcontaining one or more nucleic acids encoding a PARP, PARG, and/or ARH3protein, and/or a PARP, PARG, and/or ARH3 fusion protein. A transgenicanimal may be a mouse, a rat, a bovine, an ovine, a caprine, a porcine,a horse, a rabbit, or a monkey. The nucleic acid encoding one or morePARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3 fusionproteins may contain a tissue-specific promoter that allows theexpression of one or more transgenic proteins into a biological fluid ofthe mammal (e.g., into the milk or serum of the transgenic mammal). Forexample, a protein may be engineered for expression in the milk of amammal by placing the sequence encoding the protein downstream of thecasein promoter (U.S. Pat. No. 4,873,316). A PARP, PARG, and/or ARH3protein, and/or PARP, PARG, and/or ARH3 fusion protein produced in abiological fluid of a transgenic mammal may be purified as describedbelow.

Methods for the production of a transgenic mammal from a transgenic cellare known in the art and include, without limitation, methods thatrequire the transfer of a nucleus from a transgenic cell to anenucleated oocyte and/or the microinjection of one or more nucleic acids(e.g., a plasmid or an artificial chromosome) encoding one or more PARP,PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3 fusionproteins into an oocyte. Such genetically manipulated oocytes may thenbe transferred into a recipient female host to produce a transgenicmammal.

Cell Lysates

Cell lysates may be prepared from the transgenic cells containing anucleic acid encoding one or more PARP, PARG, and/or ARH3 proteins,and/or PARP, PARG, and/or ARH3 fusion proteins of the invention. Celllysates may be prepared by any methods known in the art, including bothphysical disruption methods and chemical disruption methods. Physicaldisruption methods include, but are not limited to sonication,homogenization, and rapid freeze/thaw lysis. Chemical disruption methodsinclude, but are not limited to, the use of lysis buffers (e.g., bufferscontaining a detergent such as Triton-X-100 and NP-40). Following lysisof the cell membrane using chemical and/or physical disruption methods,the lysate may optionally be centrifuged to remove cellular debrisand/or partially purified by one or more (e.g., 1, 2, 3, 4, 5, 6, or 7)of the following steps: salt gradient precipitation (e.g., ammoniumsulfate precipitation), size exclusion chromatography or dialysis, andcolumn chromatography (e.g., affinity chromatography, size exclusionchromatography, anion exchange chromatography, and cation exchangechromatography). The cell lysate may also be treated with one or more(e.g., 1, 2, or 3) of a DNAse, RNAse, or lipase prior to further use.One or more (e.g., 1, 2, 3, 4, or 5) protease inhibitors may also beadded to the cell lysate prior to use.

PARP, PARG, and ARH Protein and Fusion Protein Purification

One or more PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/orARH3 fusion proteins may be fully or partially purified (e.g., at least60%, 70%, 80%, 85%, 90%, 95%, and 99% pure from other proteins in thecell) from cell lysates, a biological medium (e.g., cell culture mediumfrom a transgenic cell), or a biological fluid (e.g., blood, serum, ormilk) from transgenic mammal expressing one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more)nucleic acids encoding a PARP, PARG, and/or ARH3 protein, and/or a PARP,PARG, and/or ARH3 fusion protein of the invention. Alternatively, one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 or more) PARP, PARG, and/or ARH3 proteins, and/or PARP,PARG, and/or ARH3 fusion proteins may be fully or partially purifiedfrom the extracellular medium of a transgenic cell expressing one ormore nucleic acids encoding a PARP, PARG, and/or ARH3 protein, and/or aPARP, PARG, and/or ARH3 fusion protein of the invention. In eachexample, a cell lysate, biological fluid (e.g., milk or serum), orextracellular medium containing one or more PARP, PARG, and/or ARH3proteins, and/or PARP, PARG, and/or ARH3 fusion proteins is collected.

Methods for the purification of a recombinant protein from a celllysate, biological fluid, or extracellular medium are known in the art.For example, in instances where the PARP, PARG, and/or ARH3 fusionprotein contains an epitope, an antibody specific for the epitope (e.g.,anti-GFP antibodies, anti-FLAG antibodies, anti-GST antibodies,anti-hemagglutinin antibodies, anti-c-myc antibodies, and anti-V5antibodies) may be used to purify one or more PARP, PARG, and/or ARH3fusion protein(s). In another example, a PARP, PARG, and/or ARH3 fusionprotein may contain a polypeptide tag containing a sequence that aids inaffinity purification of the protein (e.g., a His₆ tag, acalmodulin-binding protein tag, a glutathione S-transferase protein tag,a strep II tag, a HPC tag, a maltose-binding protein tag). In eachexample, a solid surface, resin, or bead (e.g., magnetic bead) may becovalently attached to a protein or molecule specifically bound by theprotein sequence located in the polypeptide tag. In such instances,contacting the one or more PARP, PARG, and/or ARH3 fusion protein(s)with the solid surface, resin, or bead will cause the selective bindingof the one or more PARP, PARG, and/or ARH3 fusion protein(s) with thesolid surface, resin, or bead. The remaining non-bound proteins will notbind and may be washed away using an appropriate buffer. Specificmethods for the affinity purification of proteins are known in the art.

One or more PARP, PARG, and/or ARH3 fusion proteins may also be purifiedfrom a cell lysate, biological sample, or a extracellular medium by apurification protocol including, but limited to: salt precipitation(e.g., ammonium sulfate precipitation), pH precipitation, precipitationusing organic solvents, high performance liquid chromatography (HPLC),column chromatography, ion exchange chromatography (e.g., cationexchange chromatography and anion exchange chromatography), immobilizedmetal affinity chromatography, gel filtration, or size exclusionchromatography or dialysis. One or more (e.g., 1, 2, 3, 4, 5, 6, or 7)of these steps may also be used in combination with an affinitypurification step as described above.

The one or more purified PARP, PARG, and/or ARH3 proteins, and/or PARP,PARG, and/or ARH3 fusion proteins may be dialyzed to exchange the bufferor concentrated prior to use in one or more of the assays describedherein (e.g., PARP, PARG, and/or ARH3 activity assays or assays for theidentification of a specific PARP activator or inhibitor). The one ormore purified PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG,and/or ARH3 fusion proteins may be stored at −70° C. in the presence orabsence of one or more (e.g., 1, 2, 3, 4, or 5) stabilizing proteinsincluding, but not limited to, albumin.

PARP Protein and PARP Fusion Protein Assays

The biological activity of the one or more PARP proteins and PARP fusionproteins of the invention include, but are not limited to, one or more(e.g., 1, 2, 3, 4, or 5) of the ability to covalently attach anADP-ribose molecule to one or more (e.g., 1, 2, 3, 4, or 5) substrate(s)(e.g., a protein, a RNA molecule, a DNA molecule, or a lipid), theability to covalently attach an ADP-ribose molecule to a ADP-riboseresidue covalently attached to a substrate, the ability to add abranched ADP-ribose molecule to a pre-existing poly-ADP-ribose, theability to localize to the cell nucleus, the ability to localize tostress granules, the ability to catalyze the formation or nucleation ofstress granules, the ability to catalyze the disassembly of stressgranules, the ability to promote cell division and mitosis, or theability to inhibit RNAi activity in the cell. Specific PARP proteinshave a different subset of biological activities: PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16 have the ability to localize tothe nucleus and/or the ability to promote cell division and mitosis;PARP5A, PARP12, PARP13.1, PARP13.2, and PARP15 have the ability tolocalize to stress granules and the ability to promote or nucleatestress granule formation; PARP11 has the ability to localize to stressgranules and the ability to promote disassembly of stress granules orprevent the formation of stress granules; and PARP13.1 has the abilityto decrease the activity of RNAi and the ability to add one or moreADP-ribose molecules to Argonaut.

Assays to measure the ability of one or more PARP proteins and PARPfusion protein(s) to covalently attach an ADP-ribose to one or more(e.g., 1, 2, 3, 4, or 5) substrate(s) (e.g., a protein, a RNA, a DNA, ora lipid) involve the incubation of one or more PARP fusion protein(s)with the one or more substrate(s) in the presence of a labeled NAD⁺molecule (e.g., radiolabeled, fluorescently-labeled, andcolorimetrically-labeled NAD⁺). A radiolabeled NAD⁺ substrate maycontain one or more radioisotopes including, but not limited to, ¹⁴C(e.g., ¹⁴C-adenine), ³²P, and ³H. Additional NAD⁺ substrates includefluorescently-labeled NAD⁺ (Putt et al., Anal. Biochem. 78:326, 2004),colorimetrically-labeled NAD⁺ (Nottbohn et al., Agnew. Chem. Int. Ed.46:2066-2069, 2007), and biotinylated NAD⁺ (6-biotin-17-NAD; R & DSystems). Following incubation of the one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) PARPproteins and/or the one or more PARP fusion proteins with the labeledNAD⁺ and one or more (e.g., 1, 2, 3, 4, or 5) substrate molecules, thespecific labeling of the substrate(s) with one or more labeledADP-ribose molecules is determined by measuring the amount of the labelassociated with the NAD⁺ that is covalently bound to the one or moresubstrate molecules. An increase (e.g., at least 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100%) in the amount of the labelassociated with the NAD⁺ that is covalently bound to the one or moresubstrate(s) indicates PARP protein or PARP fusion protein activity.

In another example of a PARP assay, the auto-modification of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 or more) PARP proteins or PARP fusion protein(s) is measuredby incubating the one or more PARP proteins and/or PARP fusion proteinsof the invention with a labeled NAD⁺ substrate and subsequently,measuring the amount of the label associated with the NAD⁺ covalentlybound to the one or more PARP fusion proteins. An increase in the amountof the label associated with the NAD⁺ covalently bound to the one ormore PARP fusion proteins indicates PARP fusion proteinauto-modification.

In an alternative assay, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) PARP proteinsand/or PARP fusion proteins may be incubated with one or more (e.g., 1,2, 3, 4, or 5) substrates and a non-labeled NAD⁺. Thepoly-ADP-ribosylation of the one or more substrates may be measured bycontacting the one or more substrates with a poly-ADP-ribose antibody.For example, a sample of substrate proteins may be electrophoresed andimmunoblotted with an anti-poly-ADP-ribose antibody. An increased numberof proteins or an increased level of detection using an anti-poly-ADPribose antibody indicates an increase in the activity of the one or morePARP fusion proteins.

Assays to measure the ability of a PARP protein and/or PARP fusionprotein to localize to a specific cellular structure or organelle usingimmunofluorescence microscopy are known in the art. For example,antibodies specific for one or more PARP proteins and/or fusionproteins, and antibodies specific for one or more proteins or moleculesspecific to a cellular structure or organelle (e.g., cytoskeleton,mitochondria, trans-Golgi network, endoplasmic reticulum, earlyendosome, centrosome, GW bodies, nuclear envelope, lysosome,peroxisomes, histones, Cajal bodies, nucleus, and mitochondria) may beused to perform immunofluorescent microscopy. Localization of one ormore PARP proteins and/or PARP fusion proteins may be measured inhigh-throughput experiments by co-localization of one or more PARPproteins and/or fusion proteins with one or more proteins specific for acellular structure or organelle (e.g., proteins listed in FIG. 10).Localization of one or more PARP proteins and/or PARP fusion proteins inthe nucleus may also be demonstrated by co-localization of a dye thatstains DNA and an antibody that specifically binds the one or more PARPproteins and/or PARP fusion proteins (e.g., co-localization of anantibody specific for one or more PARP proteins and/or PARP fusionproteins, and 4′,6-diamindino-2-phenylindole (DAPI)).

Localization of one or more PARP proteins and/or PARP fusion proteins toa specific cell structure or organelle may occur only during one or more(e.g., 1, 2, 3, 4, 5, or 6) specific stages of the cell cycle,including, but not limited to, G2-M, prophase, prometaphase (P-M),metaphase, anaphase, cytokinesis, G_(o), and G₁ stages. For the purposesdescribed herein, a PARP protein and/or PARP fusion protein is deemed tohave the ability to localize to a specific cellular structure ororganelle if it localizes to the specific cellular structure ororganelle in at least one stage (e.g., mitosis or cytokinesis) of thecell cycle.

The ability of a PARP protein and/or fusion protein to promote stressgranule assembly or to inhibit stress granule assembly may be measuredusing fluorescence microscopy. In such a method, cells are treated withone or more PARP inhibitors, one or more PARP activators, or a nucleicacid encoding one or more PARP proteins and/or PARP fusion proteins, andare subsequently fixed and immunostained with antibodies specific forone or more stress granule protein (e.g., one or more of eIF3, eIF1A,eIF2α, eIF3θ, eIF4A1, eIF4e, and G3BP). An increase in the number offoci containing one or more stress granule proteins (e.g., intenseimmunostaining in distinct cellular structures) indicates an increase inthe formation of stress granules. A decrease in the number of focicontaining one or more stress granule proteins, likewise, indicates adecrease in the formation of stress granules. In such assays, stressgranule formation may be induced by exposure to stress conditions, forexample, by treatment with sodium arsenite and pateamine A.

The ability of one of more PARP proteins and/or PARP fusion proteins topromote cell division and mitosis may be measured using any method knownin the art. For example, cell proliferation assays including, but notlimited to, standard cell counting assays, BrdU labeling, andquantitative assays for DNA synthesis such as ³H-thymidine incorporationmay be used to measure the ability of one or more PARP proteins and/orPARP fusion proteins to promote cell division and mitosis. Likewise,inhibition of one or more PARP proteins and/or PARP fusion proteins withthe ability to promote cell division and mitosis may result in celldeath. Several assays to measure cell death are known in the art,including, but not limited to Hoechst 33342 staining of chromatin,propidium iodide staining, annexin V staining of phosphoserine, and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)staining.

Assays for measuring RNAi activity in a cell are available in the art.For example, psiCHECK™-1, psiCHECK™-2, and pGL4.72[hRlucCP]™ vectorassay systems provide methods for the measurement of RNAi activity in acell. In these assays systems, luciferase is used a primary reportergene and a target sequence (i.e., the target of one or more RNAimolecules) is cloned a multiple cloning region located downstream of theluciferase translational stop codon. Initiation of the RNAi processtowards the target gene results in the cleavage and subsequentdegradation of the fusion mRNA encoded by the vectors (i.e., upontreatment of the transfected cell with a vector-target RNAi molecule).Measurement of decreased luciferase activity in the transfected cellsfollowing treatment with the vector-target RNAi indicates the activityof RNAi in the cell. For example, in experiments using the psiCHECKassay system, a cell transfected with the psiCHECK vector is treatedwith the vector-target RNAi and with an activator or inhibitor of one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP proteins and/or PARPfusion proteins (e.g., 1, 2, 3, 4, or 5 RNAi molecules targeting aspecific PARP). Transfected cells treated with the vector-target RNAiand with a PARP inhibitor or activator that demonstrate increasedluciferase activity relative to a transfected cell treated with thevector-target RNAi alone indicate that the specific targeted PARPactivates or inhibits RNAi activity in the cell, respectively. Cellstreated with a PARP inhibitor or activator that demonstrate decreasedluciferase activity relative to a cell treated with vector-target RNAialone indicate that the specific targeted PARP inhibits or activatesRNAi activity in the cell, respectively.

Any of the above-referenced PARP protein and/or PARP fusion proteinactivity assays may be performed to determine the activity of PARPprotein or PARP fusion protein sequence encoded by a nucleic acid havingat least 80% sequence identity to one of SEQ ID NOS: 1-24. The domainstructure of several PARP proteins are shown in FIG. 3. Preferredmutations in the wild-type sequences of PARP proteins (e.g., SEQ ID NOS:1-24) do not introduce amino acid changes in the catalytic domain inFIG. 3. In addition, the biological activity of a PARP protein and/orPARP fusion protein containing a sequence having at least 80% sequenceidentity to one of SEQ ID NOS: 1-24 may be assessed using any of theabove-described cellular or in vitro assays.

PARG and ARH3 Protein and Fusion Protein Assays

The activity of one or more PARG protein, ARH3 protein, PARG fusionprotein, and/or ARH3 fusion protein may be determined using assays knownin the art. PARG and ARH3 proteins herein have been demonstrated todecrease or prevent the formation of stress granules. Assays for themeasurement of stress granule formation and disassembly are describedherein.

Additional assays for PARG and ARH3 proteins (and fusion proteins)include the hydrolysis of poly-ADP-ribose. Labeled poly-ADP-ribose(e.g., ³²P, ¹⁴C, or biotinylated ADP) may be used as a substrate for themeasurement of the hydrolysis and release of ADP-ribose from a labeledand/or attached poly-ADP-ribose polymer.

Additional assays for PARG and ARH3 proteins involve the use ofantibodies specific for poly-ADP-ribose. For example, cells may betransfected with a nucleic acid that overexpresses a PARG protein, ARH3protein, PARG fusion protein, and/or ARH3 fusion protein and cellsuntreated or treated with a stress condition (e.g., sodium arsenite).Cells that contain an active form of a PARG protein, ARH3 protein, PARGfusion protein, and/or ARH3 fusion protein show decreased staining forpoly-ADP-ribose than cells transfected with a control form or inactiveform of these proteins (e.g., a form lacking its catalytic domain or aform containing an inactivating mutation).

These activity assays will also aid in the identification of which aminoacids may be mutated to generate nucleic acids having at least 80%(e.g., at least 85%, 90%, 95%, 99%, or 100% identity) to PARG (SEQ IDNO: 42) or ARH3 (SEQ ID NO: 41). Preferred sites of mutation lie outsidethe catalytic domains of PARG and/or ARH3.

PARP-Specific Antibodies

Antibodies specific to the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) PARP proteinsand/or PARP fusion proteins of the invention can be generated usingstandard methods, such as those described herein. Antibodies specificfor one or more PARP fusion proteins, PARP proteins, or fragments ofPARP proteins and/or PARP fusion proteins may be used in quantitativeassays to measure to amount of one or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or or more) PARP proteinsand/or PARP fusion proteins present in a cell, cell lysate, biologicalsample, or extracellular medium. Antibodies specific to the one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 or more) PARP proteins and/or PARP fusion proteins of theinvention may also be used to identify specific binding partners orpotential inhibitors or activators of the one or more PARP proteinsand/or PARP fusion proteins.

For the preparation of polyclonal antibodies reactive with one or morePARP proteins and/or PARP fusion proteins, one or more PARP protein(s),PARP fusion protein(s), fragments of PARP protein(s), or fragments ofPARP fusion protein(s) can be purified from natural sources (e.g.,cultures of cells expressing one or more PARP proteins) or synthesizedin, e.g., mammalian, insect, or bacterial cells by expression ofcorresponding DNA sequences contained in a suitable cloning vehicle(e.g., the nucleic acids encoding PARP proteins and PARP fusion proteinsdescribed herein). Fusion proteins are commonly used as a source ofantigen for producing antibodies. The antigenic proteins can beoptionally purified, and then coupled to a carrier protein, mixed withFreund's adjuvant to enhance stimulation of the antigenic response in aninoculated animal, and injected into rabbits, mice, or other laboratoryanimals. Primary immunizations are carried out with Freund's completeadjuvant and subsequent immunizations performed with Freund's incompleteadjuvant. Following booster injections at bi-weekly intervals, theinoculated animals are then bled and the sera isolated. The sera is useddirectly or is purified prior to use by various methods, includingaffinity chromatography employing reagents such as Protein A-Sepharose,antigen-Sepharose, and anti-horse-Ig-Sepharose. Antibody titers can bemonitored by Western blot and immunoprecipitation analyses using one ormore PARP proteins, PARP fusion proteins, and/or fragments of PARPfusion proteins or PARP proteins. Immune sera can be affinity purifiedusing one or more PARP proteins, PARP fusion proteins, and/or fragmentsof PARP fusion proteins or PARP proteins coupled to beads. Antiserumspecificity can be determined using a panel of proteins, such as one ormore PARP proteins, PARP fusion proteins, and/or fragments of PARPfusion proteins or PARP proteins.

Alternatively, monoclonal antibodies are produced by removing the spleenfrom the inoculated animal, homogenizing the spleen tissue, andsuspending the spleen cells suspended in phosphate buffered saline(PBS). The spleen cells serve as a source of lymphocytes, some of whichproduce antibody of the appropriate specificity. These cells are thenfused with permanently growing myeloma partner cells, and the productsof the fusion plated into a number of tissue culture wells in thepresence of selective agents, such as hypoxanthine, aminopterine, andthymidine (Mocikat, J. Immunol. Methods 225:185-189, 1999; Jonak et al.,Hum. Antibodies Hybridomas 3:177-185, 1992; Srikumaran et al., Science220:522, 1983). The wells can then be screened by ELISA to identifythose containing cells making antibody capable of binding to one or morePARP proteins, PARP fusion proteins, fragments of PARP proteins, and/orfragments of PARP fusion proteins, or mutants thereof. These cells canthen be re-plated and, after a period of growth, the wells containingthese cells can be screened again to identify antibody-producing cells.Several cloning procedures can be carried out until over 90% of thewells contain single clones that are positive for specific antibodyproduction. From this procedure, a stable cell line of clones thatproduce the antibody are established. The monoclonal antibody can thenbe purified by affinity chromatography using Protein A Sepharose andion-exchange chromatography, as well as variations and combinations ofthese techniques. Once produced, monoclonal antibodies are also testedfor specific PARP protein and/or PARP fusion protein recognition byELISA, Western blot, and/or immunoprecipitation analysis (see, e.g.,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981).

As an alternate or adjunct immunogen to a PARP protein and/or PARPfusion protein, peptides corresponding to relatively unique regions of aPARP protein or PARP fusion protein can be generated and coupled tokeyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine.Antiserum to each of these peptides can be similarly affinity-purifiedon peptides conjugated to BSA, and specificity tested by ELISA andWestern blotting using peptide conjugates, and by Western blotting andimmunoprecipitation using a PARP protein, PARP fusion protein, and/orfragment of a PARP protein or PARP fusion protein.

Antibodies of the invention are desirably produced using PARP proteinand/or PARP fusion protein amino acid sequences that do not residewithin highly conserved regions, and that appear likely to be antigenic,as evaluated by criteria such as those provided by the Peptide StructureProgram (Genetics Computer Group Sequence Analysis Package, ProgramManual for the GCG Package, Version 7, 1991) using the algorithm ofJameson et al., CABIOS 4:181, 1988. These fragments can be generated bystandard techniques, e.g., by PCR, and cloned into any appropriateexpression vector. For example, GST fusion proteins can be expressed inE. coli and purified using a glutathione-agarose affinity matrix. Tominimize the potential for obtaining antisera that is non-specific orexhibits low-affinity binding to one or more PARP proteins, PARP fusionproteins, and/or fragments of PARP proteins or PARP fusion proteins, twoor three PARP fusion proteins may be generated for each fragmentinjected into a separate animal. Antisera are raised by injections inseries, preferably including at least three booster injections.

In addition to intact monoclonal and polyclonal anti-PARP protein oranti-PARP fusion protein antibodies, various genetically engineeredantibodies and antibody fragments (e.g., F(ab′)2, Fab′, Fab, Fv, and sFvfragments) can be produced using standard methods. Truncated versions ofmonoclonal antibodies, for example, can be produced by recombinantmethods in which plasmids are generated that express the desiredmonoclonal antibody fragment(s) in a suitable host. Ladner (U.S. Pat.Nos. 4,946,778 and 4,704,692) describes methods for preparing singlepolypeptide chain antibodies. Ward et al., Nature 341:544-546, 1989,describes the preparation of heavy chain variable domain which have highantigen-binding affinities. McCafferty et al. (Nature 348:552-554, 1990)show that complete antibody V domains can be displayed on the surface offd bacteriophage, that the phage bind specifically to antigen, and thatrare phage (one in a million) can be isolated after affinitychromatography. Boss et al. (U.S. Pat. No. 4,816,397) describes variousmethods for producing immunoglobulins, and immunologically functionalfragments thereof, that include at least the variable domains of theheavy and light chains in a single host cell. Cabilly et al. (U.S. Pat.No. 4,816,567) describes methods for preparing chimeric antibodies. Inaddition, the antibodies can be coupled to compounds, such as toxins orradiolabels.

Methods for Identification of Specific PARP Inhibitors or Activators

The PARP proteins and/or PARP fusion proteins of the invention may beused to identify one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) specific PARP activatorsor inhibitors. In the provided assays, one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) PARPproteins and/or PARP fusion proteins are contacted with an agent (e.g.,a test agent), a labeled NAD⁺ (e.g., a colorimetrically-labeled,fluorescently-labeled, biotinylated-, or radioisotope-labeled NAD⁺), andone or more substrates, and measuring the amount of labeled ADP-ribosecovalently attached to the one or more substrates. In one example, oneor more PARP protein and/or PARP fusion proteins of the invention areincubated with a labeled NAD⁺ substrate and the amount of labelassociated with the NAD⁺ that is covalently attached to the one or morePARP proteins and/or PARP fusion proteins is measured (e.g.,auto-modulation activity assay). In this example, an agent that is aspecific PARP inhibitor mediates a decrease (e.g., at least a 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%,95%, or even 100% decrease) in the amount of labeled ADP-ribosecovalently attached to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) PARP proteins and/or fusionproteins, wherein the label on the PARP-fusion proteins is the same asthe label of the NAD⁺. In a method for identifying an agent that is aspecific PARP activator, the agent mediates an increase (e.g., at leasta 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,80%, 90%, 95%, or even 100% increase) in the amount of labeledADP-ribose covalently attached to one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) PARP proteinsand/or PARP fusion proteins.

The one or more PARP proteins and/or PARP fusion proteins utilized ineach assay may be purified, partially purified (e.g., at least 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, or 95% pure) or may be present in a celllysate (e.g., a bacterial cell lysate, a yeast cell lysate, or amammalian cell lysate), in a biological fluid from a transgenic animal(e.g., milk or serum), or an extracellular medium. The one or more PARPproteins and/or PARP fusion proteins utilized in the assay may be boundto substrate, such as, but not limited to, a solid surface (e.g., amulti-well plate), a resin, or a bead (e.g., a magnetic bead).

In additional examples of the assays, the one or more PARP proteinsand/or PARP fusion proteins may be bound to a solid surface, resin, orbead (e.g., a magnetic bead) and subsequently treated with one or moreprotease(s) (e.g., a TEV protease) prior to contacting the one or morePARP proteins and/or PARP fusion proteins with the labeled NAD⁺.

In preferred assays, an activator or inhibitor increases or decreasesthe amount of labeled ADP-ribose covalently attached to a specific PARPprotein, PARP fusion protein, and/or subset of PARP proteins or fusionproteins while having no or little (e.g., less than 50%, 40%, 30%, 25%,20%, 15%, 10%, or 5% change (e.g., increase or decrease)) affect on theamount of labeled ADP-ribose covalently attached to other PARP proteinsand/or PARP fusion proteins, is identified as a PARP activator orinhibitor, respectively. For example, the assay desirably identifies anagent that specifically inhibits the amount of labeled ADP-ribosecovalently attached to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10) PARP5A proteins or fusion proteins, PARP12 proteins and/or fusionproteins, PARP13.1 proteins and/or fusion proteins, PARP13.2 proteinsand/or fusion proteins, and PARP15 proteins and/or fusion proteins.Another assay desirably identifies an agent that specifically increasesthe amount of labeled ADP-ribose covalently attached to one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP5A proteins and/or fusionproteins, PARP12 proteins and/or fusion proteins, PARP13.1 proteinsand/or fusion proteins, PARP 13.2 proteins and/or fusion proteins, andPARP15 proteins and/or fusion proteins. Another example of the assaydesirable identifies an activator or inhibitor that specificallyincreases or decreases, respectfully, the amount of labeled ADP-ribosecovalently bound to one or more (e.g., 1, 2, 3, 4, 5, or 6) PARP11proteins and/or fusion proteins. Another example of the assay desirablyidentifies an agent that specifically decreases the amount of labeledADP-ribose covalently attached to one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10) PARP1 proteins and/or fusion proteins, PARP2 proteinsand/or fusion proteins, PARP5A proteins and/or fusion proteins, PARP5Bproteins and/or fusion proteins, PARP7 proteins and/or fusion proteins,PARP8 proteins and/or fusion proteins, PARP14 proteins and/or fusionproteins, and PARP16 proteins and/or fusion proteins. Another example ofthe assay desirably identifies an agent that specifically increases theamount of labeled ADP-ribose covalently attached to one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARP1 proteins and/or fusion proteins,PARP2 proteins and/or fusion proteins, PARP5A proteins and/or fusionproteins, PARP5B proteins and/or fusion proteins, PARP7 proteins and/orfusion proteins, PARP8 proteins and/or fusion proteins, PARP14 proteinsand/or fusion proteins, and PARP16 proteins and/or fusion proteins. Inanother desirable embodiment of the assay, the assay identifies an agentthat specifically increases or decreases the amount of labeledADP-ribose covalently attached to one or more (e.g., 1, 2, 3, 4, 5, or6) different PARP13.1 proteins and/or fusion proteins.

A variety of different agents may be tested in the above-describedassays provided by the invention. For example, a tested agent may be aderived from or present in a crude lysate (e.g., a lysate from amammalian cell or plant extract) or be derived from a commerciallyavailable chemical libraries. Large libraries of natural product orsynthetic (or semi-synthetic) extracts or chemical libraries arecommercially available and known in the art. The screening methods ofthe present invention are appropriate and useful for testing agents froma variety of sources for activity as a specific PARP activator orinhibitor. The initial screens may be performed using a diverse libraryof agents, but the method is suitable for a variety of other compoundsand compound libraries. Such compound libraries can also becombinatorial libraries. In addition, compounds from commercial sourcescan be tested, as well as commercially available analogs of identifiedinhibitors.

An agent may be a protein, a peptide, a DNA or RNA aptamer (e.g., a RNAimolecule), a lipid, or a small molecule (e.g., a lipid, carbohydrate, abioinorganic molecule, or an organic molecule).

Agents that may be tested as a specific PARP activator include nucleicacids that contain a sequence encoding one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) domains of a PARP protein (e.g., a domain encoded bypart of the nucleic acid sequence having at least 80% sequence identityto any one of SEQ ID NOS: 1-24).

Methods for Identification of an Agent that Specifically Binds One orMore PARPs

The invention also provides methods for identifying an agent thatspecifically binds to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) PARP proteins and/orPARP fusion proteins. These methods require the contacting of one ormore of the PARP proteins and/or PARP fusion proteins of the inventionwith a test agent and determining whether the test agent specificallybinds to the one or more PARP proteins and/or PARP fusion proteins. Anagent that specifically binds one or more of the described PARP proteinsand/or PARP fusion proteins may act as an activator or inhibitor of theexpression or activity of the one or more PARP proteins and/or PARPfusion proteins in a cell. For example, an agent that specifically bindsto one or more PARP proteins and/or PARP fusion proteins may selectivelyincrease the activity or expression of one or more PARP proteins and/orfusion proteins, while at the same time decreasing the activity orexpression of one or more other PARP proteins and/or PARP fusionproteins in the same cell or sample.

The one or more PARP proteins and/or PARP fusion proteins used in thismethod may be attached to a solid surface or substrate (e.g., a bead)and/or may be present in purified form or present in a crude celllysate, biological fluid, or extracellular medium. The methods mayoptionally include one or more (e.g., 1, 2, 3, 4, or 5) washing stepsfollowing contacting the one or more PARP proteins and/or PARP fusionproteins with the test agent. The test agent may be a small molecule, alipid, an RNA molecule, a DNA molecule, a protein, or a peptidefragment. The test agent may be purified in form (e.g., at least 70%,80%, 85%, 90%, 95%, or 99% pure by weight) or may be present in a crudecell lysate. The test agent may also, optionally be labeled (e.g., acolorimetric label, a radionuclide label, labeled with a biotinmolecule, or labeled with a fluorophore).

The binding of the test agent to one of more PARP proteins and/or PARPfusion proteins may detected by any known method including, BIAcore,competitive binding assays (e.g., a competitive binding assay using oneor more of the antibodies provided by the invention), and detection ofthe agent following its release from the one or more PARP proteinsand/or PARP fusion proteins (e.g., elution of the bound test agentfollowing exposure to high salt or a high or low pH buffer). The one ormore PARP proteins and/or PARP fusion proteins may be any of the examplePARP proteins and PARP fusion proteins described herein.

In one example of this method, a bead attached to one or more PARPproteins and/or PARP fusion proteins of the invention (e.g., aZZ-TEV-PARP fusion protein) may be incubated with a crude cell lysate,and the proteins or peptide fragments bound to the one or more PARPproteins and/or PARP fusion proteins may be eluted from the beads byexposure to a high salt buffer, a high detergent buffer, or a high orlow pH buffer. The resulting eluted proteins may be electrophoresed ontoan SDS-polyacrylamide gel and the specific protein bands cut out fromthe gel and analyzed using mass spectrometry to identify the specificagent that binds to the one or more PARP proteins and/or PARP fusionproteins.

In another example of the method, a bead attached to one or more PARPproteins and/or PARP fusion proteins of the invention is incubated witha purified protein or peptide fragment. In this instance, a protein orpeptide fragment bound to the one or more PARP proteins and/or PARPfusion proteins may be eluted using a high salt buffer, a high detergentbuffer, or a high or low pH buffer. The amount of protein in the eluatemay be detected by any method known in the art including UV/visspectroscopy, mass spectrometry, or any colorimetric protein dye (e.g.,a Bradford assay).

In specific screening assays for agents that bind one or more PARPproteins and/or PARP fusion proteins, one or more PARP proteins and/orPARP fusion proteins may be placed in individual wells of a multi-wellplate (e.g., one or more PARP proteins and/or PARP fusion proteinscovalently linked to the plate surface) and incubated with the testagent. Following a washing step, the amount of test agent remaining ineach well may be determined and the ability of the test agent to bindone or more PARP proteins and/or PARP fusion proteins determined.

The methods desirably identify a test agent that specifically binds oneor more of a PARP1 protein and/or fusion protein, a PARP2 protein and/orfusion protein, a PARP5A protein and/or fusion protein, a PARP5B proteinand/or fusion protein, a PARP7 protein and/or fusion protein, a PARP8protein and/or fusion protein, a PARP14 protein and/or fusion protein,and a PARP16 protein and/or fusion protein of the invention. The methodsalso desirably identify a test agent that specifically binds to one ormore of a PARP5A protein and/or fusion protein, a PARP12 protein and/orfusion protein, a PARP13.1 protein and/or fusion protein, a PARP13.2protein and/or fusion protein, and a PARP15 protein and/or fusionprotein of the invention. The methods also desirably identify a testagent that specifically binds to a PARP13.1 protein and/or fusionprotein or a PARP11 protein and/or fusion protein of the invention.

Methods for Quantification of the Level of One or More PARPs in a Sample

The present invention further provides methods for quantitating thelevel of one or more PARP proteins or PARP fusion proteins present in asample (e.g., a cell, a cell lysate, a biological fluid, or anextracellular medium). In these methods, a cell, cell lysate, biologicalfluid, or extracellular medium is contacted with one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) antibodies of the invention (e.g.,antibodies that specifically bind to one or more PARP proteins or PARPfusion proteins described herein) and the level of one or more PARPproteins and/or PARP fusion proteins is determined by measuring theamount of the one or more PARP proteins and/or PARP fusion proteinsbound to the one or more antibodies.

In these methods, the one or more antibodies may be polyclonalantibodies. The antibodies used in these methods may also be covalentlybound to a bead (e.g., a magnetic bead or a bead in a column) or may becovalently bound to the surface of a multi-well plate (e.g., for use inan enzyme-linked immunosorbent assay (ELISA)). The quantitation of thebinding of the one or more antibodies to the one or more PARP proteinsand/or PARP fusion proteins may be determined by any method known in theart, including, but not limited to, BIAcore, immunofluorescencemicroscopy, immunofluorescence-assisted cell sorting, ELISA,immunoblotting, and competitive binding assays (e.g., assays usingpurified labeled PARP proteins and/or PARP fusion proteins).

In these assays, the level of one or more PARP proteins and/or PARPfusion proteins may be compared to a standard curve control generatedusing one or more purified PARP proteins and/or PARP fusion proteins asdescribed herein. The level of one or more PARP proteins present in acell, cell lysate, or biological sample may be used as an indicator ofthe status or severity of one or more stress granule-related condition(e.g., a neurodegenerative disease, a cardiovascular disease, aninflammatory disease, and ischemia-reperfusion injury). For example, anincrease in the level of one or more of PARP5A, PARP12, PARP13.1,PARP13.2, and PARP15 indicates an increased severity or an increase inthe likelihood of developing a stress granule-related disorder.

Agent Screening Assays

The invention further provides methods for identifying a candidate agentfor treating or decreasing a stress granule-related disorder requiringthe steps of: providing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10) PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3fusion proteins encoded by a nucleic acid containing a nucleic acidsequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or even 100%) to PARP5A (SEQ ID NO: 8 or 9),PARP12 (SEQ ID NO: 18), PAPR13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO:20), PARP15 (SEQ ID NO: 22 or 23), PARG (SEQ ID NO: 42), or ARH3 (SEQ IDNO: 41); contacting the one or more PARP, PARG, and/or ARH3 proteins,and/or PARP, PARG, and/or ARH3 fusion proteins with the agent and alabeled NAD⁺ substrate; and measuring the activity of the one or morePARP, PARG, and/or ARH3 proteins and/or PARP, PARG, and/or ARH3 fusionproteins or the specific binding of the agent to the one or more PARPfusion proteins; wherein an agent that decreases (e.g., at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%) the activity ofthe one or more PARP proteins and/or one or more PARP fusion proteins,and/or specifically binds to the one or more PARP proteins or fusionproteins, and/or increases the activity of the one or more PARG and/orARH3 proteins or fusion proteins is identified as an agent for treatingor decreasing the likelihood of developing a stress granule-relateddisorder.

The invention further provides methods for identifying a candidate agentfor treating or decreasing the likelihood of developing cancer requiringthe steps of: providing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10) PARP proteins and/or PARP fusion proteins encoded by a nucleic acidcontaining a nucleic acid sequence having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) to aPARP selected from PARP1 (SEQ ID NO: 1 or 2), PARP2 (SEQ ID NO: 3),PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ ID NO: 10), PARP7 (SEQ ID NO:12), PARP8 (SEQ ID NO: 13), PARP14 (SEQ ID NO: 21), or PARP16 (SEQ IDNO: 24); contacting the one or more PARP proteins and/or PARP fusionproteins with the agent and a labeled NAD⁺ substrate; and measuring theactivity of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) PARPproteins and/or PARP fusion proteins; wherein an agent that decreases(e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even100%) the activity and/or specifically binds to the one or more PARPproteins and/or PARP fusion proteins is identified as a candidate agentfor treating or decreasing the likelihood of developing cancer.

In all the above screening methods, the one or more PARP, PARG, and/orARH3 proteins, and/or PARP, PARG, and/or ARH3 fusion proteins may begenerated using any methods known in the art or the methods describedherein. The PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/orARH3 fusion proteins may be purified or present in a crude cell lysate.The PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3fusion proteins may be attached to a substrate (e.g., a magnetic bead)or a solid surface (multi-well plate).

The methods of the assay include the measurement of the activity of theone or more PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/orARH3 fusion proteins using any assay known in the art or those assaysdescribed herein. The assays may include the incubation of the one ormore PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3fusion proteins with a labeled NAD⁺ substrate and/or the incubation withother substrates that are not labeled (e.g., non-labeled NAD⁺). EachPARP, PARG, and/or ARH3 protein, and/or PARP, PARG, and ARH3 fusionprotein may have one or more activities (described herein). Any knownactivity of a PARP, PARG, and/or ARH3 protein, and/or PARP, PARG, and/orARH3 fusion protein (or activity described herein) may be measured inthe above screening assays (e.g., catalytic addition of an ADP-ribosemolecule or polymerization of a poly-ADP-ribose molecule, catalyticdegradation of poly-ADP ribose or removal of a ADP-ribose moiety from asubstrate, localization to a particular cell structure (e.g., nucleus orstress granule), formation of a stress granule, disassembly of a stressgranule, nucleation of a stress granule, promotion of cell proliferationor cell division, or required for cell cycle progression (e.g.,progression through cytokinesis or mitosis)).

The ability of an agent to bind to one or more PARP or PARP fusionproteins may be measured using any method known in the art, as well asthose methods described herein. For example, the ability of an agent tospecifically bind to a PARP protein or PARP fusion protein may bemeasured by chromatography (e.g., using beads wherein a PARP protein, aPARP fusion protein, or a domain of a PARP protein or PARP fusionprotein is covalently attached), BIAcore analysis, UV/vis spectroscopy,or calorimetry.

A variety of different agents may be tested in the above-describedscreening assays provided by the invention. For example, a candidateagent may be a derived from or present in a crude lysate (e.g., a lysatefrom a mammalian cell or plant extract) or be derived from acommercially available chemical libraries. Large libraries of naturalproduct or synthetic (or semi-synthetic) extracts or chemical librariesare commercially available and known in the art. The screening methodsof the present invention are appropriate and useful for testing agentsfrom a variety of sources for activity as a candidate agent. The initialscreens may be performed using a diverse library of agents, but themethod is suitable for a variety of other compounds and compoundlibraries. Such compound libraries can also be combinatorial libraries.In addition, compounds from commercial sources can be tested, as well ascommercially available analogs of identified inhibitors.

An agent may be a protein, a peptide fragment, a DNA or RNA aptamer(e.g., a RNAi molecule), a lipid, or a small molecule (e.g., a lipid,carbohydrate, a bioinorganic molecule, or an organic molecule).

Candidate agents that may be tested include nucleic acids that contain asequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)domains of a PARP protein (e.g., a domain encoded by part of the nucleicacid sequence having at least 80% sequence identity to any one of SEQ IDNOS: 1-24), nucleic acids that contain a sequence encoding one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) domains of a PARG protein orARH3 (e.g., a domain encoded by part of the nucleic acid sequence havingat least 80% sequence identity to SEQ ID NO: 41 and 42), or anypolypeptide encoded by these nucleic acids.

In any of the above screening assays, the NAD⁺ may be labeled with aradioisotope (e.g., ³²P) or fluorophore (e.g., fluorescein), or bebiotinylated.

Methods for Increasing Cell Proliferation in a Cell

The invention further provides methods of increasing the proliferationof a cell or population of cells requiring contacting the cell orpopulation of cells with an effective amount of one or more PARPactivators. In these methods, the one or more (e.g., 1, 2, 3, 4, or 5)PARP activators preferably increase the expression (e.g., mRNA and/orprotein) and/or one or more activities of a PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16. In these methods, theexpression may be an increase (e.g., at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100%) in the level of one or more nucleic acidsequence(s) containing a sequence having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100%) to PARP1(SEQ ID NOS: 1 or 2), PARP2 (SEQ ID NO: 3), PARP5A (SEQ ID NOS: 8 or 9),PARP5B (SEQ ID NO: 10), PARP14 (SEQ ID NO: 21), or PARP16 (SEQ ID NO:24), or one or more polypeptides encoded by one of these nucleic acids.The activity of one or more PARPs may be any activity described herein,including, but not limited to, poly-ADP-ribosylation of a target protein(e.g., a protein localized to the nucleus or mitotic spindle duringcytokinesis, or a PARP protein) or is required for progression throughmitosis or cytokinesis.

A PARP activator encompassed by these methods may include one or morenucleic acid sequences containing a sequence having at least 80%sequence identity (e.g., 85%, 90%, 95%, 99%, or even 100%) to one of SEQID NOS: 1-3, 8-10, 21, or 24.

The rate of proliferation may be increased in a mammalian cell or aplant cell using these methods. For example, the rate of proliferationof a primary cell (e.g., a cell used for cell replacement therapies) maybe increased using these methods. In another example, the rate ofproliferation of a plant cell may be increased using these methods.These methods include the introduction of xenogenous nucleic acidsencoding a PARP activator to create a transgenic mammalian or plant cellor transgenic mammal or plant.

Methods for Increasing Stress Granule Formation in a Cell

The invention further provides methods of increasing (e.g., by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the number ofstress granules in a cell or cell population requiring contacting thecell or population of cells with one or more (e.g., 1, 2, 3, 4, or 5)PARP activators, one or more (e.g., 1, 2, 3, 4, or 5) PARG inhibitors,or one or more (e.g., 1, 2, 3, 4, or 5) PARP11 inhibitors. In thesemethods, the increased number of stress granules may be the result of anincrease in the rate of stress granule nucleation or formation and/or adecrease in the rate of stress granule breakdown or turnover.

In these methods, the one or more PARP activators preferably increase(e.g., at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%) the expression (e.g., mRNA and/or protein) and/or activity of aprotein encoded by a nucleic acid sequence containing a sequence that isat least 80% identical (e.g., at least 85%, 90%, 95%, 99%, or even 100%identical) to PARP5A (SEQ ID NO: 8 or 9), PARP12 (SEQ ID NO: 18),PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO: 20), or PARP15 (SEQ IDNO: 22 or 23). The activity of the one or more PARPs may be an increasein the poly-ADP-ribosylation of one or more (e.g., 1, 2, 3, 4, or 5)target protein(s) (e.g., a protein localized in a stress granule, aprotein involved in the formation or disassembly of a stress granule,and/or a PARP protein) or the nucleation and/or formation of a stressgranule. Additional activities of a PARP protein are described herein.Examples of the PARP activators that may be used with these methodsinclude, nucleic acid sequence containing a sequence having at least 80%identity (e.g., at least 85%, 90%, 95%, 99%, or even 100% identical) toany one of SEQ ID NOS: 8, 9, 18-20, 22, or 23.

In these methods, the one or more PARG inhibitors may selectivelydecrease (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100% decrease) in the expression (e.g., mRNA and/or protein) oractivity of PARG or glycohydrolase ARH3. For example, one or more PARGinhibitors may decrease the level of one or more nucleic acidsequence(s) containing a sequence having at least 80% sequence identity(e.g., at least 85%, 90%, 95%, 99%, or even 100% identity) to PARG (SEQID NO: 42) or ARH3 (SEQ ID NO: 41), or a polypeptide encoded by thesenucleic acids. The activities of PARG protein or ARH3 glycohydrolase aredescribed herein and include, without limitation, the hydrolysis ofpoly-ADP-ribose (e.g., poly-ADP-ribose attached to a protein, e.g., aprotein localized in a stress granule, a protein involved in theformation or disassembly of a stress granule, and/or a PARP), theprevention of the assembly of a stress granule, or disassembly of astress granule. Examples of PARG inhibitors that may be used in thesemethods include an antibody that specifically binds to a polypeptide orpeptide fragment encoded by a nucleic acid sequence containing asequence having at least 80% sequence identity (e.g., 85%, 90%, 95%,99%, or even 100%) to PARG (SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41), oran RNA aptamer that binds to one or more of these nucleic acidsequences. Specific examples of RNAi molecules that may target PARG andARH3 are SEQ ID NOS: 34 and 35, and SEQ ID NOS: 36 and 37, respectively.

In these methods, the one or more PARP11 inhibitors may selectivelydecrease (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, oreven 100%) the expression (mRNA and/or protein) and/or activity ofPARP11. In different examples of these methods, the PARP11 inhibitor(s)may decrease the expression of one or more nucleic acid(s) containing asequence having at least 80% sequence identity (e.g., at least 85%, 90%,95%, 99%, or even 100%) to PARP11 (SEQ ID NO: 17), or decrease theexpression of one or more polypeptides encoded by these nucleic acids.The activity of PARP11 may include poly-ADP-ribosylation of a targetprotein (e.g., a protein localized in a stress granule, a polypeptideinvolved in the formation or disassembly of a stress granule, and/or aPARP), prevention of the assembly of a stress granule, or disassembly ofa stress granule. Examples of PARP11 inhibitors that may be used inthese methods include an antibody that specifically binds to apolypeptide or peptide fragment encoded by a nucleic acid sequencecontaining a sequence having at least 80% sequence identity (e.g., 85%,90%, 95%, 99%, or even 100%) to PARP11 (SEQ ID NO: 17), or an RNAaptamer that binds to one or more of these nucleic acid sequences.Specific examples of RNAi molecules that may target PARP11 are SEQ IDNOS: 91-98.

These methods may allow for the specific up-regulation of stress granuleformation in cells in which increased resistance to toxic stress isdesired (e.g., a plant cell (e.g., a transgenic plant cell) or acultured mammalian cell (e.g., for cell replacement therapies).

EXAMPLES

The features and other details of the invention will now be moreparticularly described and pointed out in the following examplesdescribing preferred techniques and experimental results. These examplesare provided for the purpose of illustrating the invention and shouldnot be construed as limiting.

Example 1 Generation of PARP-GFP Fusion Proteins and Assays

Fusion proteins containing the sequence of each PARP and greenfluorescent protein (GFP) were generated using the pEGFP-C1 vector(Invitrogen) (FIG. 4). For these experiments, the DNA sequences encodingeach of PARP1 (SEQ ID NOS: 1 and 2), PARP3 (SEQ ID NOS: 4, 5, and 6),PARP4 (SEQ ID NO: 7), PARP5A (SEQ ID NOS: 8 and 9), PARP5B (SEQ ID NO:10), PARP6 (SEQ ID NO: 11), PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO:13), PARP9 (SEQ ID NO: 14), PARP10 (SEQ ID NOS: 15 and 16), PARP11 (SEQID NO: 17), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2(SEQ ID NO: 20), PARP14 (SEQ ID NO: 21), PARP15 (SEQ ID NOS: 22 and 23),and PARP16 (SEQ ID NO: 24) were cloned into the pEGFP-C1 vector usingthe restriction sites indicated in Table 2. Each resulting plasmidcontained a nucleic acid sequence encoding a PARP-GFP fusion protein,wherein the nucleic acid sequence encoding GFP was located 5′ to thenucleic acid sequence encoding the PARP protein.

TABLE 2 Restriction Sites Used for Cloning PARP Sequences into pEGFP-C1PARP Restriction Sites  1 BglII, SalI  3 BglII, SalI  4 KpnI, ApaI  5aHinDIII, BglII  5b SalI, BamHI  6 SalI, XmaI  7 BspEI, EcoRI  8 BspEI,SalI  9 BspEI, SalI 10 BamHI, BglII 11 SalI, BamHI 12 SalI, ApaI 13isoform 1 BspEI, BamHI 13 isoform 2 BglII, BamHI 14 XhoI, SacII 15 SalI,BamHI 16 BglII, SalI

Each generated pEGFP-C1 vector was transfected into HeLa Kyoto cellsusing Lipofectamine 2000, according to the manufacturer's instructions.Cell lysate was prepared from the HeLa cells at 48 hours followingtransfection. Electrophoresis was performed on the cell lysate using4-12% SDS-PAGE, and immunoblotting was performed using a rabbit anti-GFPpolyclonal antibody (FIG. 5).

The localization of each PARP-GFP fusion protein in the transfected HeLaKyoto cells was determined using immunofluorescence microscopy usingrabbit anti-GFP polyclonal antibody and a fluorescently-labeledsecondary antibody (FIG. 6). The data from this experiment indicate thatseveral PARP-GFP proteins are primarily localized in the nucleus ofasynchronous cells, including PARP1-GFP, PARP2-GFP, PARP7-GFP, andPARP8-GFP. The data further indicate that several PARP-GFP fusionproteins are localized in primarily in the cytoplasm of asynchronouscells, including PARP12-GFP, PARP13-GFP, PARP14-GFP, PARP15-GFP,PARP16-GFP, PARP10-GFP, PARP11-GFP, PARP5A-GFP, and PARP5B-GFP. Inaddition, the data indicate that several PARP-GFP fusion proteins arelocalized in both the cytoplasm and the nucleus of asynchronous cells,including PARP3-GFP, PARP9-GFP, PARP6-GFP, and PARP4-GFP. The samepattern of cell localization for each PARP-GFP fusion protein wasobserved in the hTERT-RPE1 cell line (Clontech), atelomerase-immortalized human retinal pigment epithelial (RPE) normalcell line (FIG. 7).

Antibodies specific for each PARP were generated by immunizing rabbitswith PARP-specific peptides conjugated to keyhole limpet hemocyanin(KLH) using known methods. The antibodies produced in the rabbit serumwere later affinity purified using peptide columns (e.g., columnscontaining, as substrate, the specific peptide sequence used toinoculate the rabbit).

The antibodies for each PARP and a secondary-fluorescently labeledanti-rabbit polyclonal antibody were used to visualize the location ofeach PARP in asynchronous HeLa Kyoto cells transfected with a pEGFP-C1plasmid encoding a PARP-GFP fusion protein (FIG. 8). The data from thisexperiment confirm that PARP1, PARP2, PARP7, PARP8, and PARP14 areprimarily localized in the nucleus of asynchronous cells. The data fromthis experiment also confirm that PARP3, PARP4, PARP6, PARP9, and PARP15are localized in the both the nucleus and the cytoplasm of asynchronouscells.

The localization of each PARP-GFP fusion protein (described above) wasalso determined in HeLa Kyoto cells transfected with a pEGFP-C1 plasmidencoding a PARP-GFP fusion protein following 12-hour treatment with 100nM nocodazole or 5 μg/mL aphidicolin. Cells treated with nocodazole arearrested in S phase, while cells treated with aphidicolin are arrestedin mitotosis. FIG. 9 shows the cellular localization for each PARP-GFPfusion protein following cell arrest in S-phase or mitosis. The datashow that the PARP1-GFP, PARP2-GFP, and PARP8-GFP fusion proteinslocalize to the nucleus during S-phase, and that PARP5A-GFP andPARP5B-GFP localize to the mitotic spindle during mitosis. Thelocalization of these PARP-GFP fusion proteins (e.g., PARP1-GFP,PARP2-GFP, PARP5A-GFP, PARP5B-GFP, and PARP8-GFP) to the nucleus duringS-phase and mitosis indicate a role for these PARP proteins in celldivision and cell proliferation.

In order to study the role of PARP16, additional experiments wereperformed using RNAi knockdown of endogenous PARP16 or overexpression ofPAPR16-GFP fusion proteins to study the effect of PARP16 knockdown andoverexpression, respectively on cell morphology. Asynchronous HeLa Kyotocells overexpressing PARP16-GFP protein had normal cell morphology (FIG.10; middle panel). In these cells, the PARP16-GFP protein was primarilylocalized in the endoplasmic reticulum, as demonstrated by itsco-localization with calnexin (FIG. 10; middle panel). HeLa Kyoto cellstransfected with an RNAi molecule specific for PARP16 demonstratedsignificant morphological changes, including cell shrinkage and dramaticmembrane defects (FIG. 10; right panel).

The specific cellular localization of each PARP-GFP fusion protein maybe further analyzed by immunofluorescence microscopy using a combinationof labeled antibodies specific for the GFP-tag of each PARP-GFP fusionprotein and one or more markers of cellular structures or organelles.For example, immunofluorescence staining of asynchronous HeLa Kyotocells transfected with a pEGFP-C1 vector expressing the PARP7-GFP fusionprotein shows co-localization of an anti-GFP antibody and an anti-coilinantibody (a marker of Cajal bodies in the nucleus) (FIG. 11). In anotherexample, asynchronous HeLa Kyoto cells transfected with a pEGFP-C1vector expressing the PARP16-GFP fusion protein shows co-localization ofan anti-GFP antibody and an anti-calnexin antibody (a marker of theendoplasmic reticulum) (FIG. 10). A non-limiting list of marker proteinsthat may be used to determine the cellular localization of a PARP-GFPfusion protein is also provided in FIG. 11.

Experimental Methods

Kyoto HeLa cells were grown in DMEM supplemented with 10% FCS andpenicillin/streptomycin at 37° C. in 5% CO₂. Lipofectamine 2000(Invitrogen) was used to transfect the cells with each pEGFP-C1 vectoraccording to the manufacturer's protocol. Cells were arrested in mitosisand S-phase by treatment with 100 nM nocodazole or 5 μg/mL aphidicolinfor 12 hours, respectively. For immunofluorescence imaging, cells oncoverslips were fixed in ice-cold methanol for five minutes andrehydrated in phosphate buffered saline (PBS). The cells were blocked inPBS containing 4% bovine serum albumin (BSA) and 0.1% Triton-X 100. Allantibodies used for imaging were diluted in blocking buffer. Thecoverslips were incubated with primary antibodies for 45 minutes andwith secondary antibodies for 30 minutes. Images were collected on aNikon TE2000 confocal microscope equipped with a Yokogawa CSU-X1spinning disk head, Hamamatsu ORCA ER digital camera, and NIS-Elementsimaging software.

Example 2 Generation of ZZ-TEV-PARP Fusion Proteins

Fusion proteins containing the sequence of each PARP, a ZZ-domain of SEQID NO: 27, and four TEV protease recognition sequences (SEQ ID NO: 26)were cloned using the pcDNA3.1 vector (SEQ ID NO: 33) (Invitrogen) (FIG.12) to yield a ZZ-4x-TEV-PARP fusion protein for each PARP. For theseexperiments, the DNA sequences encoding PARP1 (SEQ ID NOS: 1 and 2),PARP2 (SEQ ID NO: 3), PARP3 (SEQ ID NOS: 4, 5, and 6), PARP4 (SEQ ID NO:7), PARP5A (SEQ ID NOS: 8 and 9), PARP6 (SEQ ID NO: 11), PARP7 (SEQ IDNO: 12), PARP9 (SEQ ID NO: 14), PARP10 (SEQ ID NOS: 15 and 16), PARP11(SEQ ID NO: 17), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQ ID NO: 20),PARP14 (SEQ ID NO: 21), PARP15 (SEQ ID NOS: 22 and 23), and PARP16 (SEQID NO: 24) were cloned into the pcDNA3.1 vector using the restrictionsites indicated in Table 3. The sequence encoding the ZZ-domain and thesequence encoding the four TEV protease recognition sequences werecloned into the NheI and HinDIII restriction sites in pcDNA3.1.

TABLE 3 Restriction Sites Used for Cloning PARP Sequences into pcDNA3.1PARP Restriction Sites  1 XhoI, PmeI  2 BamHI, NotI  3 EcoRV, NotI  4KpnI, ApaI  5a HinDIII, XhoI  6 EcoRV, NotI  7 BamHI, NotI  9 EcoRV,NotI 10 HinDIII, XbaI 11 BamHI, XbaI 13 isoform 1 KpnI, BamHI 13 isoform2 BamHI, EcoRV 14 KpnI, XhoI 15 KpnI, XhoI 16 KpnI, XbaI

Each resulting plasmid contained a nucleic acid sequence encoding aZZ-TEV-PARP fusion protein, wherein the nucleic acid sequence encodingZZ domain was located 5′ to the nucleic acid sequence encoding the fourTEV protease recognition sequences, which in turn, was located 5′ to thenucleic acid sequence encoding each PARP.

Nucleic acids encoding each ZZ-TEV-PARP fusion protein may betransfected into target cells (e.g., HeLa Kyoto or HeLa S3 cells) andthe resulting ZZ-TEV-PARP fusion proteins purified by binding tomagnetic beads coated with a protein containing an Fc domain (e.g.,IgG). The resulting ZZ-TEV-PARP fusion proteins may be used in theassays described below for the PARP-GFP fusion proteins and the otherassays described herein. Assays utilizing the ZZ-TEV-PARP fusionproteins have the additional advantage of containing an engineered TEVprotease recognition sequence, whereby the polypeptide tag on each PARPfusion protein (e.g., the ZZ-domain and the four TEV proteaserecognition sequences) may optionally be removed from the ZZ-TEV-PARPfusion proteins by treatment with TEV protease. In one example, one ormore ZZ-TEV-PARP fusion proteins may be removed from a magnetic bead,resin, or solid surface by treatment with a TEV protease.

Example 3 PARP Activity Assays and Screening Methods

The above-described PARP fusion proteins may be used in PARP activityassays and in assays to identify an activator or inhibitor for aspecific PARP or a specific subset of PARPs. An example of such anactivity assay in shown in FIG. 13. In this example, cell lysate isfirst prepared from a HeLa S3 cell culture expressing one or morePARP-GFP fusion proteins. The cell lysate is then incubated with ananti-GFP polyclonal antibody bound to Dynabead® Protein A beads, and thebeads magnetically removed from the cell lysate. The isolated beadsbound to one or more PARP-GFP fusion proteins are placed into amulti-well plate and incubated with a labeled NAD⁺ substrate (e.g.,³²P-NAD⁺). Following incubation with the labeled NAD⁺ substrate, themagnetic beads bound with the one or more PARP-GFP proteins aremagnetically isolated or washed, and the level of the label (i.e., thelabel present in the labeled NAD⁺ substrate) that is covalently attachedto the one or more PARP-GFP fusion proteins bound to the magnetic beadsis determined (e.g., the amount of ³²P covalently bound to the one ormore PARP-GFP proteins attached to the beads). This assay provides ameans of measuring the auto-modulation activity of one or more PARP-GFPfusion proteins (e.g., the ability of a PARP to modify its own structureby catalyzing the covalent attachment of one or more ADP-ribosemolecules). The assay may also be designed such that lysate or PARP-GFPfusion proteins isolated from several different transfected HeLa S3cells, each expressing a different PARP-GFP fusion proteins or subset ofPARP-GFP fusion proteins, may be placed in different wells of themulti-well plate. The assay may also be designed such that the lysatefrom several different transfected HeLa S3 cells is combined, whereinthe lysate from each transfected HeLa S3 cell culture contains one ormore PARP-GFP fusion proteins. In a different version of the assay, thePARP-GFP fusion proteins may contain a protease recognition site. Inthis version of the assay, the one or more PARP-GFP fusion proteinsbound to the magnetic beads may be treated with a specific protease(i.e., a protease that recognizes a protease recognition sequence in thePARP-GFP fusion protein) to mediate release of the PARP-GFP fusionprotein from the magnetic bead.

FIG. 14 provides an example of the use of the PARP-GFP fusion proteinsof the invention for the identification of an agent that specificallyinhibits the activity of one or more PARPs. This assay is similar to theassay described above, except that the one or more PARP-GFP fusionproteins is incubated with both a test agent and a labeled NAD⁺substrate. A specific PARP inhibitor will decrease the amount of thelabel (i.e., the label present in the labeled NAD⁺ substrate) covalentlyattached to the one or more PARP-GFP fusion proteins bound to themagnetic beads relative to the amount of the label attached to the oneor more PARP-GFP fusion proteins in the absence of the test agent. Indifferent examples of this assay, lysate or PARP-GFP fusion proteinsisolated from two or more different transfected HeLa S3 cells, eachexpressing a different PARP-GFP fusion protein or subset of PARP-GFPfusion proteins, may be placed in different wells of the multi-wellplate. The assay may also be designed such that the lysate from severaldifferent transfected HeLa S3 cells is combined, wherein the lysate fromeach transfected HeLa S3 cells contains one or more PARP-GFP fusionproteins. The assay may also be specifically designed to identifyinhibitors of a specific PARP-GFP protein or subset of PARP-GFP proteinsincluding the subsets of: one or more of PARP1-GFP, PARP2-GFP,PARP5A-GFP, PARP5B-GFP, PARP7-GFP, PARP8-GFP, PARP14-GFP, andPARP16-GFP; one or more of PARP5A-GFP, PARP12-GFP, PARP13.1-GFP,PARP13.2-GFP, PARP15-GFP; PARP11-GFP; or PARP13.1-GFP.

Similar to the examples, described above, the PARP-GFP fusion proteinsof the invention may be used to identify activators of one or morespecific PARPs. In this instance, the assay may be used to identifyagents that increase the amount of the label (i.e., the label present inthe labeled NAD⁺ substrate) covalently attached to the one or morePARP-GFP fusion proteins bound to the magnetic beads relative to theamount of the label covalently attached to the one or more PARP-GFPfusion proteins in the absence of the test agent. Preferably, this assaymay be designed to identify activators of a specific PARP-GFP fusionprotein or subset of PARP-GFP fusion proteins including the subsets of:one or more of PARP1-GFP, PARP2-GFP, PARP5A-GFP, PARP5B-GFP, PARP7-GFP,PARP8-GFP, PARP14-GFP, and PARP16-GFP; one or more of PARP5A-GFP,PARP12-GFP, PARP13.1-GFP, PARP13.2-GFP, and PARP15-GFP; PARP11-GFP; orPARP13.1-GFP.

Example 4 Involvement of PARPs in Stress Granule Formation andDisassembly

We have discovered through a PARP family-wide RNAi screen that severalPARP proteins are involved in the cell cycle and are required forprogression through mitosis (e.g., PARP16). The identity of the varioussubstrate proteins of the different PARP proteins remains largelyunknown. To further identify PARP substrate proteins and/or proteinsthat bind to poly-ADP-ribose polymers, the Bio-Gel P-6 resin shown inFIG. 15 was used to purify proteins that bind poly-ADP-ribose polymerand/or act as an acceptor of a ADP-ribose molecule or a poly-ADP-ribosepolymer. FIG. 15 also shows a Coomassie Blue-stained SDS-PAGE gelshowing the proteins present in the HeLa Kyoto cell extract (Extract),in cell extract following lectin clarification (Lectin Clarification),in the lysate prior to passing over the Bio-Gel P-6 resin (Input), inthe pellet following centrifugation of the resin (Pellet), and in theeluate following treatment with poly-ADP-ribose glycohydrolase ARH3(ARH3 Release). The data in FIG. 15 demonstrates the selectivepurification of proteins bound to the Bio-Gel P-6 resin.

We have discovered that poly-ADP-ribose polymers are associated withstress granules in cells during exposure to stress conditions. FIG. 16shows the co-localization of poly-ADP-ribose polymers and eIF3, a markerof stress granules, in HeLa Kyoto cells following treatment with 0 or250 μM sodium arsenite for 30 minutes and immunostaining withfluorescently-labeled antibodies specific for poly-ADP-ribose polymersand eIF3. The data indicate that stress granules contain proteinsmodified with poly-ADP-ribose polymers.

In order to identify the specific PARP proteins that mediate theformation of the poly-ADP-ribose polymers present in stress granules,experiments were performed to determine whether the different PARP-GFPfusion proteins localize to stress granules. In these experiments, HeLaKyoto cells transfected with a pEGFP-C1 plasmid expressing a PARP-GFPfusion protein were visualized using fluorescently-labeled anti-GFP andanti-eIF3 antibodies following treatment with 250 μM sodium arsenite for30 minutes (FIG. 17). The data indicate that the PARP5A-GFP, PARP12-GFP,PARP13.1-GFP, PARP13.2-GFP, and PARP15-GFP fusion proteins localize tostress granules under stress conditions.

Endogenous PARP5A, PARP12, PARP13/13.1, PARP15, and poly-ADP-riboseglycohydrolase (PARG) also localize to stress granules in HeLa Kyotocells following treatment with 250 μM sodium arsenite for 30 minutes(FIG. 18). In these experiments, the fixed cells were visualized usingantibodies specific for one of PAR5A, PARP12, PARP13/13.1, PARP15, orPARG, and an anti-eIF3 antibody, and secondary fluorescently-labeledantibodies. The data indicate that PARG, as well as the endogenous- andfusion protein-forms of PARP5A, PARP12, PARP13/13.1, and PARP15,localize to stress granules under stress conditions. In a similar set ofexperiments using hTERT RPE cells, endogenous PARP5A, PARP12, PARP13,and PARP15 showed a similar cellular localization following exposure to250 μM sodium arsenite for 30 minutes, as was observed in HeLa Kyotocells (FIG. 19).

Experiments using time-lapse immunofluorescence microscopy in live HeLaKyoto cells further indicate that endogenous PARP12, PARP12-GFP,endogenous PARP13, and PARP13-GFP localize to stress granules at anearly point in stress granule assembly and therefore, may play aregulatory role in the formation of stress granules (data not shown).

In an additional set of experiments, the effect of PARP13.1, PARP13.2,and PARP15 on stress granule formation was further studied by measuringthe effect of overexpression of PARP13.1-GFP, PARP13.2-GFP, andPARP15-GFP on stress granule formation. In these experiments, HeLa Kyotocells were transfected with a pEGFP-C1 plasmid encoding PARP13.1-GFP,PARP13.2-GFP, or PARP15-GFP. The transfected cells were stained withanti-GFP antibodies, anti-eIF3 antibodies, and fluorescently-labeledsecondary antibodies. These data indicate that overexpression ofPARP13.1-GFP, PARP13.2-GFP, or PARP15-GFP fusion protein nucleatesstress granule formation (FIG. 20).

In contrast to the effect mediated by overexpression of PARP13.1-GFP,PARP13.2-GFP, or PARP15-GFP fusion protein, overexpression of PARP11-GFPin HeLa Kyoto cells mediates a decrease in stress granule formationfollowing treatment with 250 μM sodium arsenite for 30 minutes (FIG.21). In this experiment, HeLa Kyoto cells transfected with a pEGFP-C1vector expressing a PARP11-GFP fusion protein were treated with sodiumarsenite, and stained with anti-GFP antibodies, anti-eIF3 antibodies,and fluorescently-labeled secondary antibodies. These data indicate thatoverexpression of PARP11-GFP suppresses the formation of stress granulesin cells exposed to stress conditions.

Experimental Methods

HeLa Kyoto cells were cultured as described above. Lipofectamine 2000(Invitrogen) was used to transfect the HeLa Kyoto cells with a pEGFP-C1plasmid encoding a PARP-GFP fusion protein (described above) accordingto the manufacturer's instructions. For stress granule induction, cellswere treated with 250 μM sodium arsenite for 30 minutes. For long-term,real-time imaging of PARP-GFP transfected HeLa cells, the cells weresplit into 24-well glass bottom plates and imaged every 20 minutes for48 hours. Images were collected on a Nikon TE2000 confocal microscopeequipped with a Yokogawa CSU-X1 spinning disc head, Hamamatsu ORCA ERdigital camera, and NIS-Elements imaging software.

Example 5 Involvement of PARG and ARH3 in Stress Granule Disassembly

In order to determine the importance of poly-ADP-ribose polymers onstress granule formation and disassembly, an additional set ofexperiments were performed to test the effect of PARG and ARH3 activityon stress granule dynamics. In a first set of experiments, HeLa Kyotocells were transfected with a pEGFP-C1 plasmid encoding PARG99-GFP,PARG102-GFP, or a PARG110-GFP fusion protein. Overexpression ofPARG99-GFP, PARG102-GFP, or PARG110-GFP reduces the formation of stressgranules in HeLa Kyoto cells following exposure to 100 μM sodiumarsenite for 30 minutes (FIG. 22). In these experiments, formation ofstress granules was determined by staining the fixed cells withanti-eIF3 antibodies and secondary fluorescently-labeled antibodies.These data indicate that PARG activity (hydrolysis of poly-ADP-ribose)inhibits the formation of stress granules in cells under stressconditions.

Another set of experiments was performed to determine the effect ofknockdown of PARG (SEQ ID NO: 42) or ARH3 (SEQ ID NO: 41) on stressgranule formation in cells under stress conditions. In theseexperiments, HeLa Kyoto cells were treated with 30 nM siRNA specific forPARG (SEQ ID NOS: 34 and 35) or ARH3 (SEQ ID NOS: 36 and 37), or acontrol siRNA (All Stars Negative Control siRNA; Qiagen Catalog No.1027280), and treated with 100 μM sodium arsenite for 30 minutes, or 30minutes or 1 hour following sodium arsenite washout (FIG. 23). Cellstreated with a PARG siRNA or ARH3 siRNA show a sustained presence ofstress granules following removal of sodium arsenite from the culturemedium (via imaging using anti-eIF3 antibodies and fluorescently-labeledsecondary antibodies). These data indicate that PARG and ARH3 activity(hydrolysis of poly-ADP-ribose) has a positive effect on stress granuledisassembly, and that poly-ADP-ribose turnover kinetics regulate theformation/disassembly of stress granules. The percentage of cells withstress granules following 30-minute washout and 1-hour washout afterarsenite treatment was quantitated for cells treated with control siRNA,PARG siRNA, and ARH3 siRNA (FIG. 24). These data indicate that knockdownof PARG and ARH3 reduces the rate of stress granule disassemblyfollowing removal of the stress condition (sodium arsenite).

Experimental Methods

HeLa Kyoto cells were cultured in medium as described above. In PARGoverexpression experiments, Lipofectamine 2000 (Invitrogen) was used totransfect HeLa Kyoto cells with pEGFP-C1 plasmids containing the nucleicacid sequences for each PARG isoform, i.e., PARG99, PARG102, and PARG110(sequences described in Meyer-Ficca et al., Exp. Cell. Res.297(2):521-532, 2004) according to the manufacturer's instructions. InPARG knockdown experiments, cells were treated with 30 nM of a siRNAtargeting PARG (SEQ ID NOS: 34 and 35), a siRNA targeting ARH3 (SEQ IDNO: 36 and 37), or a control siRNA (AllStars Negative Control siRNA;Qiagen Catalog No. 1027280) using Lipofectamine 2000 according to themanufacturer's instructions. In these experiments, stress granuleformation was induced by treatment with 100 μM sodium arsenite for 30minutes. For stress granule disassembly experiments, the media wasreplaced after sodium arsenite treatment, and cells were incubated for30 minutes and 1 hour prior to fixation and immunostaining. At least 200cells were counted for each condition (in triplicate) to determine thepercentage of cells containing stress granules.

Example 6 Stress Granule Proteins Bind to GFP-PARPs

Experiments were performed to further identify stress granule-relatedproteins that may bind and be the substrates of one or more of the PARPslocalized in stress granules (e.g., PARP5A, PARP12, PARP13, PARP13.1,and PARP15). In these experiments, HeLa S3 cells were transfected withpEGFP-C1 plasmids containing a nucleic acid sequence encodingPARP5A-GFP, PARP12-GFP, PARP13-GFP, PARP13.1-GFP, or PARP15-GFP fusionprotein and treated with 0 or 250 μM sodium arsenite for 30 minutes. Theresulting cell lysate was immunoprecipitated using anti-GFP antibodiesand the resulting immunoprecipitated proteins were electrophoresed usingSDS-PAGE. The resulting gel indicates that each PARP-GFP fusion proteinbinds to several proteins and that treatment with sodium arseniteresults in an alteration in the amount and identity of the proteinsbinding to each PARP-GFP fusion protein (FIG. 25A). In a similarexperiment, the immunoprecipitated proteins are transferred to amembrane and immunostained with an anti-poly-ADP-ribose antibody. Thedata in this experiment show that PARP5A-GFP, PARP12-GFP, PARP13-GFP,and PARP13.1-GFP fusion proteins bind to poly-ADP-ribosylated proteins(FIG. 25B).

Data from a separate set of experiments indicate that several stressgranule-associated proteins bind to the PARP13-GFP, PARP12-GFP, andPARP5A-GFP fusion proteins. In these experiments, HeLa S3 cells weretransfected with a pEGFP-C1 plasmid encoding a PARP13-GFP, PARP12-GFP,or PARP5A-GFP fusion protein and treated with 0 or 20 nM pateamine A for30 minutes. Cell lysates from the cells were immunoprecipitated using ananti-GFP antibody and the immunoprecipitated proteins wereelectrophoresed using 4-12% SDS-PAGE. The resulting proteins weretransferred to a membrane and immunoblotted using commercially-availableantibodies specific for different stress granule-associated proteins:Ago2, DDX6, LSM1, PABP, FMRP, eIF1A, eIF2α, eIF3ρ, eIF4A1, and eIF4E.The data indicate that the PARP13-GFP, PARP12-GFP, and PARP5A-GFP fusionproteins have the ability to interact with one or more of these stressgranule-associated proteins under both normal (0 nM pateamine A) andstress conditions (30 nM pateamine A) (FIG. 25 C).

An additional set of experiments was performed to determine whether oneor more stress granule-associated proteins are poly-ADP-ribosylated. Inthese experiments, HeLa S3 cells were transfected with a pEGFP-C1plasmid encoding a GFP fusion protein of TIA1, PABP, G3BP, or Ago2, andtreated with 0 or 20 nM pateamine A for 30 minutes. Lysates from thesecells were immunoprecipitated using anti-GFP antibodies andimmunoblotted using an anti-poly-ADP ribose antibody. The data show thatseveral proteins bind the TIA1-GFP, PABP-GFP, G3BP-GFP, and Ago2-GFPfusion proteins in untreated (0 nM pateamine A) and treated (20 nMpateamine A) cells (FIG. 26A). In an additional experiment, the proteinsthat bind to endogenous G3BP and Ago2 proteins in 250 μM sodiumarsenite-treated HeLa S3 cells were also shown to bepoly-ADP-ribosylated (FIG. 26B). In this experiment, cell lysates fromuntransfected HeLa S3 cells treated with 0 or 250 μM sodium arsenite for60 minutes were immunoprecipitated with anti-G3BP or anti-Ago2antibodies and immunoblotted using an anti-poly-ADP-ribose antibody.

G3BP1, a stress granule-associated protein, was shown to bepoly-ADP-ribosylated (FIG. 26C). In order to map the specific domain inG3BP1 that is modified by a poly-ADP-ribose polymer, GFP-fusion proteinsof different truncation 250 μM sodium arsenite for 60 minutes. Thespecific nucleic acid sequences encoding each G3BP1 truncation mutantare described in Tourriere et al. (J. Cell Biol. 160:823-831, 2003). Thecell lysate from each cell sample was immunoprecipitated using anti-GFPantibodies and immunoblotted using an anti-poly-ADP-ribose antibody. Thedata demonstrate that poly-ADP-ribosylation of G3BP1 occurs within theRNA-recognition motif (RRM) domain (“D” in FIG. 26C). The RRM domain ofG3BP1 is a domain that binds to RNA molecules. The poly-ADP-ribosylationof G3BP1 in the RRM domain is thought to regulate the RNA-bindingactivity of G3BP1.

TIA1, a stress granule-associated protein, was also shown to bepoly-ADP-ribosylated (FIG. 26D). In order to determine whether TIA1 ispoly-ADP-ribosylated in its RRM domain, GFP-fusion proteins offull-length TIA1 and a truncation mutant of TIA1 lacking its RRM domain(TIA1ΔRRM) were expressed in HeLa S3 cells treated with 0 or 250 μMsodium arsenite for 60 minutes. The specific nucleic acid sequencesencoding the full-length TIA1 and the TIA1ΔRRM truncation mutant aredescribed in Kedersha et al. (J. Cell Biol. 151:1257-1268, 2000). Thecell lysate from each cell sample was immunoprecipitated using anti-GFPantibodies and immunoblotting was performed using ananti-poly-ADP-ribose antibody. The data demonstrate thatpoly-ADP-ribosylation of TIA1 also occurs within its RNA-recognitionmotif (RRM) domain (FIG. 26D). The poly-ADP-ribosylation of TIA1 in itsRRM domain is also thought to mediate an alteration in its RNA-bindingactivity.

Experimental Methods

Immunoprecipitation experiments to identify proteins binding toPARP5A-GFP, PARP12-GFP, PARP13-GFP, PARP13.1-GFP, and PARP15-GFP wereperformed using HeLa S3 cells transfected with a pEGFP-C1 plasmidcontaining a nucleic acid sequence encoding each respective PARP-GFPfusion protein following treatment with 0 or 20 nM pateamine A for 30minutes. In each experiment, the cell lysate is incubated with ananti-GFP antibody to immunoprecipitate proteins bound to each of thePARP-GFP fusion proteins using standard methods. The resultingimmunoprepitated proteins were electrophoresed on 4-12% SDS-PAGE gels,and either stained directly with Coomassie Blue or transferred onto amembrane and immunostained with one or more of the following antibodies:anti-poly-ADP-ribose, anti-Ago2, anti-DDX6, anti-LSM1, anti-PABP,anti-FMRP, anti-eIF1A, anti-eIF2α, anti-eIF3η, anti-eIF4A1, andanti-eIF4e antibodies.

Immunoprecipitation experiments using TIA1-GFP, PABP-GFP, G3BP-GFP, andAgo2-GFP fusion proteins were performed using HeLa S3 cells transfectedwith pEGFP-C1 plasmids containing a sequence encoding a nucleic acidsequence encoding TIA1 (Kedersha et al., J. Cell Biol. 151:1257-1268,2000), PABP (NCBI Accession No. NM_(—)12154.2), G3BP (Tourriere et al.,J. Cell Biol. 160:823-831, 2003), Ago2 (NCBI Accession No._(—)002568.3),a truncation mutant of G3BP (i.e., A, ABC, BC, BCD, and D truncationmutants described in Tourriere et al., supra), or the ΔRRM truncationmutation of TIA1 (described in Kedersha et al., supra) followingtreatment with 0 or 20 nM pateamine A for 30 minutes. In eachexperiment, the cell lysate is incubated with an anti-GFP antibody toimmunoprecipitate proteins bound to each of the GFP fusion proteinsusing standard methods. The resulting immunoprepitated proteins wereelectrophoresed on 4-12% SDS-PAGE gels, and either stained directly withCoomassie Blue or transferred onto a membrane and immunostained withanti-poly-ADP-ribose antibody.

Example 7 PARP13 and PARG Regulation of RNAi Activity

We have further discovered that PARP13 and PARG regulate the activity ofRNAi and miRNA molecules in cells. Regulation of RNAi and miRNA activityin cells remains largely uncharacterized. One of the proteins implicatedfor a role in the regulation of RNAi and miRNA activity is Argonaut 2, asingle-stranded RNAse. Using immunofluorescence microscopy we haveobserved that Argonaut 2 localizes to stress granules in HeLa cellstreated with 250 μM sodium arsenite for 30 minutes (FIG. 27, leftpanel). In these experiments, cells were treated with sodium arseniteand stained using both antibodies against Argonaut 2 and eIF3 (a stressgranule marker), and secondary fluorescently-labeled antibodies. Thedata show that Argonaut 2 is poly-ADP-ribosylated in HeLa cellsfollowing exposure to 250 μM sodium arsenite for 30 minutes (FIG. 27,right panel). In these experiments, cell lysate from cells treated with250 μM sodium arsenite was immunoprecipitated with an anti-Argonaut 2antibody, and the resulting immunoprecipitated proteins wereimmunoblotted using an anti-poly-ADP-ribose antibody. The resultsindicate that Argonaut 2 is localized to stress granules andpoly-ADP-ribosylated under cellular stress conditions.

To determine whether one or more of the PARPs identified herein mediatethe poly-ADP-riboyslation of Argonaut 2, immunoprecipitation experimentswere performed on cell lysate from untransfected HeLa cells treated with0 or 250 μM sodium arsenite for 30 minutes using an anti-Argonaut 2antibody. The resulting immunoprecipitated proteins were immunoblottedusing an anti-PARP13/13.1 antibody. The data show that PARP13/13.1 bindsto Argonaut 2 under both normal (0 μM sodium arsenite) and stressconditions (250 μM sodium arsenite) (FIG. 28).

To identify additional substrate proteins of PAPR13, immunoprecipitationexperiments were performed on lysate from HeLa cells transfected with apEGFP-C1 plasmid containing a sequence encoding a PARP13-GFP fusionprotein following treatment with either 0 or 250 μM sodium arsenite for30 minutes. The cell lysate was treated with an anti-GFP antibody andthe resulting immunoprecipitated proteins were electrophoresed usingSDS-PAGE. The data show that exposure to 250 μM sodium arseniteincreases the number and identity of proteins that bind to thePARP13-GFP fusion protein (FIG. 29). The identification of the specificproteins co-immunoprecipitated with the PARP13-GFP fusion protein willhelp to further elucidate the role of PARP13 in cellular mechanisms,including its regulation of Argonaut 2 and its role in the regulation ofmiRNA and RNAi activity. Additional experiments were performed todetermine the effect of PARP13 knockdown on miRNA activity. For theseexperiments, the pGL4.72[hRlucCP]™-vector assay (Promega) was used tomeasure RNAi activity in 293T cells. The pGL4.72[hRlucCP]™ vectorcontains a constitutively expressed firefly luciferase gene which islocated upstream of several nucleic acid sequences targeted by an RNAimolecule. An increase in the activity of an RNAi molecule targeting thedownstream 3′ sequences of the vector results in a decrease in theamount of luciferase produced from the vector. In experiments to studythe effect of PARP13 on miRNA activity, the pGL4.72[hRlucCP]™ vector wasfirst engineered to contain 6 repeats of a sequence recognized by anRNAi molecule targeting the vector (“vector-target RNAi;” SEQ ID NOS: 38and 39; GUUUUCACUCCAGCUAACACA and TTCAAAAGUGAGGUCGAUUGU, respectively).In a first experiment, cells were transfected with a modifiedpGL4.72[hRlucCP]™ vector and a pEGFP-C1 plasmid encoding EGFP (negativecontrol), G3BP (negative control), PARP5a, PARP12, PARP13, PARP13.1, orPARP15; and 10 nM of the vector-target RNAi. In a positive control, thecells were transfected with the modified pGL4.72[hRlucCP]™ vector andvector-target RNAi alone (CXCR4 sponge). Cells overexpressing PARP13 orPARP13.1 showed a 3-fold decrease in the level of miRNA-mediatedrepression compared to control cells (e.g., EGFP- andG3BP-overexpressing cells) (FIG. 30). In a second set of experiments,the ability of the vector-target RNAi to reduce the expression ofluciferase was measured in 293T cells transfected with pGL4.72[hRlucCP]™vector, 20 nM vector-target RNAi, and 20 nM of negative RNAi control forPARP13 siRNA (siNeg; AllStars Negative Control siRNA; Qiagen Catalog No.1027280) or PARP13 siRNA (siPARP13; SEQ ID NO: 40) following treatmentwith 0 or 30 nM pateamine A for 2 hours. The data in FIG. 31 show thatknockdown of PARP13 expression by siPARP13 results in an increase in theactivity of the vector-target RNAi under stress conditions (i.e., 30 nMpateamine A) (FIG. 31). These data indicate that PARP13 activity in thecell has a negative effect on RNAi activity in the cell. This effect onRNAi activity may occur through the poly-ADP-ribosylation of Argonaut 2by PARP13 or by the ability of PARP13 to modify or bind other proteinslocated within stress granules or proteins required for the assembly ordisassembly of stress granules.

Experimental Methods

Immunopreciptation experiments were performed using non-transfected HeLacells following treatment with 0 or 250 μM sodium arsenite for 60minutes using an anti-Argonaut 2 antibody. The resulting precipitatedproteins were electrophoresed using 4-12% SDS-PAGE and immunoblottedusing an anti-poly-ADP ribose antibody. Non-transfected HeLa cellstreated with 250 μM sodium arsenite for 30 minutes were also stained forimmunofluorescence microscopy using antibodies specific for Argonaut 2and eIF3 (a marker of stress granules), and a secondaryfluorescently-labeled antibody (Alexa Fluor 594 and 488 antibodies).

Additional co-immunoprecipitation experiments were performed usingmethods known in the art. In these experiments, HeLa cell lysate wasprepared from cells treated with 0 or 250 μM sodium arsenite for 30minutes, and the lysate subsequently immunoprecipitated with ananti-Argonaut 2 antibody. The resulting precipitated proteins wereimmunoblotted using an anti-PARP13/13.1 antibody.

Experiments to identify additional proteins bound to a PARP13-GFP fusionprotein were performed by transfecting HeLa S3 cells with a pEGFP-C1plasmid encoding a PARP13-GFP fusion protein. The transfected cells weretreated with 0 or 250 μM sodium arsenite for 30 minutes before lysis.The resulting cell lysate was immunoprecipitated with an anti-GFPantibody and the resulting precipitated proteins were electrophoresedusing 4-12% SDS-PAGE and the resulting gel stained with Coomassie Blue.

Experiments to determine the effect of knockdown of PARP13 on miRNA andRNAi activity were performed using a modified pGL4.72[hRlucCP]™-vectorassay (Promega). For these experiments, the pGL4.72[hRlucCP]™-vector wasmodified by placing six copies of a target sequence at a position 3′ tothe luciferase gene. RNAi molecules targeting the vector were designedto bind the six copies of the target sequence (SEQ ID NOS: 34 and 35).The modified pGL4.72[hRlucCP]™-vector was introduced into 293T cellsusing Lipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. In each experiment, the cells were further transfected(Lipofectamine 2000) with 10 or 20 nM of the vector-target RNAi alone orin combination with either 20 nM of a control RNAi molecule for thesiRNA targeting PARP13 (siNeg) or an RNAi molecule targeting PARP13(siPARG13), and the cells treated with 0 or 30 nM pateamine A for 60minutes. Following 48-hours incubation, the level of luciferase proteinproduction was measured using a luciferase assay kit (Promega). The dataare shown as the relative level of luciferase protein produced in cellstransfected with the modified vector alone in the absence of any RNAitreatment.

Experiments were also performed to determine the effect ofoverexpression of a PARP-GFP protein on the activity of miRNA using themodified pGL4.72[hRlucCP]™-vector assay described above. In theseexperiments, 293T cells were transfected with pEGFP-C1 expressionvectors encoding EGFP, G3BP, PARP5A, PARP12, PARP13, PARP13.1, orPARP15; the modified pGL4.72[hRlucCP]™ vector, and 10 nMvector-targeting RNAi. As a positive control for RNAi activity, thecells were transfected with the modified pGL4.72[hRlucCP]™ and thevector-target RNAi alone (CXCR4 sponge).

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention; can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions.

1. A method of treating or decreasing the likelihood of developing astress granule-related disorder in a subject comprising administering tosaid subject a therapeutically effective amount of one or morepoly-ADP-ribose polymerase (PARP) inhibitor(s), one or more poly-ADPribose glycolase (PARG) activators, and/or one or more PARP11activators.
 2. The method of claim 1, wherein said one or more PARPinhibitor(s) selectively decrease the expression and/or one or moreactivities of one or more PARP(s) selected from the group consisting ofPARP5a, PARP12, PARP13 isoform 1 (PARP13.1), PARP13 isoform 2(PARP13.2), and PARP15.
 3. The method of claim 1, wherein said decreasein expression is a decrease in the level of one or more nucleic acid(s)comprising a nucleic acid sequence having at least 95% sequence identityto PARP5a (SEQ ID NO: 8 or 9), PARP5b (SEQ ID NO: 10), PARP13.1 (SEQ IDNO: 19), PARP13.2 (SEQ ID NO: 20), or PARP15 (SEQ ID NOS: 22 or 23), ora decrease in the level of one or more polypeptides encoded by said oneor more nucleic acid(s); or said one or more activities of said one ormore PARP(s) is poly-ADP-ribosylation of a target protein localized in astress granule or a target protein involved in the formation ordisassembly of a stress granule, or the formation or nucleation of astress granule. 4.-6. (canceled)
 7. The method of claim 1, wherein saidone or more PARG activators selectively increase the expression and/orone or more activities of PARG protein or poly-ADP-ribose glycohydrolaseARH3.
 8. The method of claim 7, wherein said increase in expression isan increase in the level of one or more nucleic acid(s) comprising anucleic acid sequence having at least 95% sequence identity to PARG (SEQID NO: 42) or ARH3 (SEQ ID NO: 41), or an increase in the level of oneor more polypeptide(s) encoded by said one or more nucleic acid(s); orsaid one or more activities of PARG or ARH3 is hydrolysis ofpoly-ADP-ribose, the prevention of the assembly of a stress granule, ordisassembly of a stress granule. 9.-12. (canceled)
 13. The method ofclaim 1, wherein said one or more PARP11 activators selectively increasethe expression and/or one or more activities of PARP11.
 14. The methodof claim 13, wherein said increase in expression is an increase in thelevel of one or more nucleic acid(s) comprising a nucleic acid sequencehaving at least 95% sequence identity to PARP11 (SEQ ID NO: 17), or anincrease in the level of one or more polypeptide(s) encoded by said oneor more nucleic acid(s); or said one or more activities of PARP11 ispoly-ADP-ribosylation of a target protein localized in a stress granuleor a target protein involved in the formation or disassembly of a stressgranule, the prevention of the assembly of a stress granule, ordisassembly of a stress granule. 15.-17. (canceled)
 18. The method ofclaim 1, wherein said one or more PARP inhibitors is an antibody or anantibody fragment that selectively binds one or more of PARP5a, PARP12,PARP13.1, PARP13.2, and PARP15 polypeptide, or an RNA aptamer, said oneor more PARG activators is one or more nucleic acid(s) comprising anucleic acid sequence having at least 95% sequence identity to PARG (SEQID NO: 42) or ARH3 (SEQ ID NO: 41); or said one or more PARP11activators is one or more nucleic acid(s) comprising a nucleic acidsequence having at least 95% sequence identity to PARP11 (SEQ ID NO:17). 19.-29. (canceled)
 30. A method of decreasing the number of stressgranules present in a cell or a population of cells comprisingcontacting said cell or population of cells with an effective amount ofone or more poly-ADP-ribose polymerase (PARP) inhibitor(s), one or morepoly-ADP ribose glycolase (PARG) activators, and/or one or more PARP11activators. 31.-51. (canceled)
 52. A method of treating or decreasingthe likelihood of developing cancer in a subject comprisingadministering to said subject a therapeutically effective amount of oneor more poly-ADP-ribose polymerase (PARP) inhibitor(s). 53.-64.(canceled)
 65. A method of decreasing proliferation of a cell or apopulation of cells by contacting said cell or population of cells withan effective amount of one or more poly-ADP-ribose polymerase (PARP)inhibitor(s). 66.-74. (canceled)
 75. A pharmaceutical compositioncomprising one or more PARP inhibitors, one or more PARG activators,and/or one or more PARP11 activators, wherein said one or more PARPinhibitor(s) selectively decrease the expression and/or one or moreactivities of one or more PARP(s) selected from the group consisting ofPARP5a, PARP12, PARP13 isoform 1 (PARP13.1), PARP13 isoform 2(PARP13.2), and PARP15. 76.-96. (canceled)
 97. A kit comprising one ormore pharmaceutical compositions of claim 75 and instructions foradministering the one or more compositions to a subject diagnosed with astress granule-related disorder or diagnosed as having a propensity todevelop a stress granule-related disorder.
 98. A pharmaceuticalcomposition comprising one or more poly-ADP-ribose polymerase (PARP)inhibitor(s), wherein said one or more PARP inhibitor(s) selectivelydecrease the expression and/or one or more activities of one or morePARP(s) selected from the group consisting of PARP1, PARP2, PARP5A,PARP5B, PARP7, PARP8, PARP14, and PARP16. 99.-106. (canceled)
 107. A kitcomprising one or more pharmaceutical compositions of claim 98 andinstructions for administering the one or more compositions to a subjectdiagnosed with cancer or diagnosed as having a propensity to develop acancer.
 108. A method for increasing the number of stress granules in acell or a population of cells comprising contacting said cell orpopulation of cells with one or more poly-ADP-ribose polymerase (PARP)activators(s), one or more poly-ADP ribose glycolase (PARG) inhibitors,and/or one or more PARP11 inhibitors. 109.-132. (canceled)
 133. A methodof increasing proliferation of a cell or a population of cellscomprising contacting said cell or population of cells with an effectiveamount of one or more poly-ADP-ribose polymerase (PARP) activators(s).134.-141. (canceled)
 142. A method of identifying a candidate agent fortreating or decreasing the likelihood of developing a stressgranule-related disorder comprising the steps of: a) providing one ormore PARP, PARG, and/or ARH3 proteins, and/or PARP, PARG, and/or ARH3fusion proteins encoded by a nucleic acid comprising a nucleic acidsequence having at least 95% sequence identity to PARP5A (SEQ ID NO: 8or 9), PARP12 (SEQ ID NO: 18), PARP13.1 (SEQ ID NO: 19), PARP13.2 (SEQID NO: 20) PARP15 (SEQ ID NO: 22 or 23), PARG (SEQ ID NO: 42), or ARH3(SEQ ID NO: 41); b) contacting said one or more PARP, PARG, and/or ARH3proteins, and/or PARP, PARG, and/or ARH3 fusion proteins with said agentand a labeled nicotinamide adenine dinucleotide (NAD⁺) substrate; and c)measuring one or more activities of said one or more PARP, PARG, and/orARH3 proteins, and/or PARP, PARG, and/or ARH3 fusion proteins, and/orthe specific binding of said agent to said one or more PARP proteins orfusion proteins; wherein an agent that decreases one or more activitiesand/or specifically binds to said one or more PARP proteins and/or PARPfusion proteins, and/or increases one or more activities of said one ormore PARG and/or ARH3 proteins, and/or PARG and/or ARH3 fusion proteinsis identified as a candidate agent for treating or decreasing thelikelihood of developing a stress granule-related disorder. 143.-149.(canceled)
 150. A method of identifying a candidate agent for treatingor decreasing the likelihood of developing cancer comprising the stepsof: a) providing one or more PARP proteins and/or PARP fusion proteinsencoded by a nucleic acid comprising a nucleic acid sequence having atleast 95% sequence identity to a PARP selected from PARP1 (SEQ ID NO: 1or 2), PARP2 (SEQ ID NO: 3), PARP5A (SEQ ID NO: 8 or 9), PARP5B (SEQ IDNO: 10) PARP7 (SEQ ID NO: 12), PARP8 (SEQ ID NO: 13), PARP14 (SEQ ID NO:21), or PARP16 (SEQ ID NO: 24); b) contacting said one or more PARPproteins and/or PARP fusion proteins with said agent and a labelednicotinamide adenine dinucleotide (NAD⁺) substrate; and c) measuring oneor more activities of said one or more PARP proteins and/or PARP fusionproteins or the specific binding of said agent to said one or more PARPproteins and/or PARP fusion proteins; wherein an agent that decreasesone or more activities and/or specifically binds to said one or morePARP proteins and/or fusion proteins is identified as a candidate agentfor treating or decreasing the likelihood of developing cancer.151.-157. (canceled)
 158. A method for determining the propensity of asubject to develop a stress granule-related disorder comprisingdetermining the expression and/or one or more activities of one or moreof PARP5A, PARP11, PARP12, PARP13.1, PARP13.2, PARP15, PARG, and ARH3 ina subject, wherein an increase in the expression and/or one or moreactivities of one or more of PARP5A, PARP12, PARP13.1, PARP13.2, andPARP15, and/or a decrease in the expression or one or more activities ofPARP11, PARG, or ARH3 indicates an increased propensity to develop astress granule-related disorder. 159.-163. (canceled)
 164. A method fordetermining the propensity of a subject to develop cancer comprisingdetermining the expression and/or one or more activities of one or moreof PARP1, PARP2, PARP5A, PARP5B, PARP7, PARP8, PARP14, and PARP16 in asubject, wherein an increase in the expression and/or one or moreactivities of one or more of PARP1, PARP2, PARP5A, PARP5B, PARP7, PARP8,PARP14, and PARP16 indicates an increased propensity to develop cancer.165. (canceled)
 166. (canceled)